Benzoimidazolone-carboxamide compounds as 5-HT4 receptors agonists

ABSTRACT

The invention provides novel benzoimidazolone-carboxamide 5-HT 4  receptor agonist compounds. The invention also provides pharmaceutical compositions comprising such compounds, methods of using such compounds to treat diseases associated with 5-HT 4  receptor activity, and processes and intermediates useful for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/688,048 filed on Jun. 7, 2005, the disclosure of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to benzoimidazolone-carboxamide compounds which are useful as 5-HT₄ receptor agonists. The invention is also directed to pharmaceutical compositions comprising such compounds, methods of using such compounds for treating or preventing medical conditions mediated by 5-HT₄ receptor activity, and processes and intermediates useful for preparing such compounds.

2. State of the Art

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that is widely distributed throughout the body, both in the central nervous system and in peripheral systems. At least seven subtypes of serotonin receptors have been identified and the interaction of serotonin with these different receptors is linked to a wide variety of physiological functions. There has been, therefore, substantial interest in developing therapeutic agents that target specific 5-HT receptor subtypes.

In particular, characterization of 5-HT₄ receptors and identification of pharmaceutical agents that interact with them has been the focus of significant recent activity. (See, for example, the review by Langlois and Fischmeister, J. Med. Chem. 2003, 46, 319-344.) 5-HT₄ receptor agonists are useful for the treatment of disorders of reduced motility of the gastrointestinal tract. Such disorders include irritable bowel syndrome (IBS), chronic constipation, functional dyspepsia, delayed gastric emptying, gastroesophageal reflux disease (GERD), gastroparesis, post-operative ileus, intestinal pseudo-obstruction, and drug-induced delayed transit. In addition, it has been suggested that some 5-HT₄ receptor agonist compounds may be used in the treatment of central nervous system disorders including cognitive disorders, behavioral disorders, mood disorders, and disorders of control of autonomic function.

Despite the broad utility of pharmaceutical agents modulating 5-HT₄ receptor activity, few 5-HT₄ receptor agonist compounds are in clinical use at present.

Accordingly, there is a need for new 5-HT₄ receptor agonists that achieve their desired effects with minimal side effects. Preferred agents may possess, among other properties, improved selectivity, potency, pharmacokinetic properties, and/or duration of action.

SUMMARY OF THE INVENTION

The invention provides novel compounds that possess 5-HT₄ receptor agonist activity. Among other properties, compounds of the invention have been found to be potent and selective 5-HT₄ receptor agonists. In addition, compounds of the invention have been found to exhibit favorable pharmacokinetic properties which are predictive of good bioavailability upon oral administration.

Accordingly, the invention provides a compound of formula (I):

wherein:

R¹ is hydrogen or C₁₋₃alkyl;

R² is hydrogen or C₁₋₃alkyl;

R³ is C₁₋₃alkyl;

R⁴ is —C(O)R⁵, —S(O)₂R⁶,

or R³ and R⁴ together with the nitrogen atom to which they are attached form

R⁵ is hydrogen, C₁₋₃alkyl, —NH₂, or pyridinyl, wherein C₁₋₃alkyl is optionally substituted with hydroxy;

R⁶ is C₁₋₃alkyl, —NH₂, or imidazolyl, wherein imidazolyl is optionally substituted with C₁₋₃alkyl;

R⁷ is —NR⁹S(O)₂C₁₋₃alkyl, —NR¹⁰C(O)R¹¹, or

R⁸ is —S(O)₂C₁₋₃alkyl or —C(O)R¹²;

R⁹, R¹⁰, and R¹¹ are each independently C₁₋₃alkyl; and

R¹² is hydrogen, C₁₋₃alkyl, or tetrahydrofuranyl;

or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof.

The invention also provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically-acceptable carrier.

The invention also provides a method of treating a disease or condition associated with 5-HT₄ receptor activity, e.g. a disorder of reduced motility of the gastrointestinal tract, the method comprising administering to the mammal, a therapeutically effective amount of a compound of the invention.

Further, the invention provides a method of treating a disease or condition associated with 5-HT₄ receptor activity in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising a compound of the invention and a pharmaceutically-acceptable carrier.

The compounds of the invention can also be used as research tools, i.e. to study biological systems or samples, or for studying the activity of other chemical compounds. Accordingly, in another of its method aspects, the invention provides a method of using a compound of formula (I), or a pharmaceutically acceptable salt or solvate or stereoisomer thereof, as a research tool for studying a biological system or sample or for discovering new 5-HT₄ receptor agonists, the method comprising contacting a biological system or sample with a compound of the invention and determining the effects caused by the compound on the biological system or sample.

In separate and distinct aspects, the invention also provides synthetic processes and intermediates described herein, which are useful for preparing compounds of the invention.

The invention also provides a compound of the invention as described herein for use in medical therapy, as well as the use of a compound of the invention in the manufacture of a formulation or medicament for treating a disease or condition associated with 5-HT₄ receptor activity, e.g. a disorder of reduced motility of the gastrointestinal tract, in a mammal.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE shows a powder x-ray diffraction (PXRD) pattern of a crystalline hydrochloride salt of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(acetylmethylamino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]yl}amide of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel benzoimidazolone-carboxamide 5-HT₄ receptor agonists of formula (I), or pharmaceutically-acceptable salts or solvates or stereoisomers thereof. The following substituents and values are intended to provide representative examples of various aspects of this invention. These representative values are intended to further define such aspects and are not intended to exclude other values or limit the scope of the invention.

In specific aspects of the invention, R¹ is ethyl, propyl, or isopropyl; or R¹ is ethyl or isopropyl.

In another specific aspect, R¹ is isopropyl.

In a specific aspect, R² is hydrogen, methyl, or ethyl.

In another specific aspect, R² is hydrogen.

In a specific aspect, R¹ is ethyl or isopropyl; and R² is hydrogen.

In specific aspects, R³ is methyl or ethyl; or R³ is methyl.

In a specific aspect, R⁴ is —C(O)R⁵.

In another specific aspect, R⁴ is —S(O)₂R⁶.

In another specific aspect, R⁴ is

In another specific aspect, R⁴ is

In another specific aspect, R⁴ is —C(O)H, —C(O)CH₃, —C(O)NH₂, —S(O)₂CH₃, —S(O)₂-1-methylimidazol-4-yl, or

In a specific aspect, R³ and R⁴ together with the nitrogen atom to which they are attached form

In another specific aspect, R³ and R⁴ together with the nitrogen atom to which they are attached form

In a specific aspect, R⁵ is hydrogen, C₁₋₃alkyl, —NH₂ or pyridinyl. In specific aspects, R⁵ is hydrogen or C₁₋₃alkyl; or R⁵ is hydrogen or methyl.

In a specific aspect, R⁶ is C₁₋₃alkyl, or imidazolyl, wherein imidazolyl is substituted with C₁₋₃alkyl. In another specific aspect, R⁶ is methyl or 1-methylimidazol-4-yl.

In yet another specific aspect, R⁶ is 1-methylimidazol-4-yl.

In specific aspects, R⁷ is —NR⁹S(O)₂CH₃ or —NR¹⁰C(O)CH₃; or R⁷ is —NR¹⁰C(O)CH₃.

In a specific aspect, R⁸ is —S(O)₂C₁₋₃alkyl, such as —S(O)₂CH₃.

In another specific aspect, R⁸ is —C(O)R¹², such as R⁸ is —C(O)H, —C(O)CH₃, or —C(O)-tetrahydrofuran-2-yl.

In a specific aspect, R⁸ is —S(O)₂CH₃, —C(O)CH₃, or —C(O)-tetrahydrofuran-2-yl.

In specific aspects, R⁹ is methyl or ethyl; or R⁹ is methyl.

In specific aspects, R¹⁰ is methyl or ethyl; or R¹⁰ is methyl.

In specific aspects, R¹¹ is methyl or ethyl; or R¹¹ is methyl.

In a specific aspect, R¹² is methyl or tetrahydrofuranyl.

In yet another aspect, the invention provides a compound of formula (I) wherein:

R¹ is ethyl or isopropyl;

R² is hydrogen;

R³ is methyl;

R⁴ is —C(O)H, —C(O)CH₃, —C(O)NH₂, —S(O)₂CH₃, —S(O)₂-1-methylimidazol-4-yl, or

or R³ and R⁴ together with the nitrogen atom to which they are attached form

R⁸ is —S(O)₂CH₃, —C(O)CH₃, or —C(O)-tetrahydrofuran-2-yl.

The chemical naming conventions used herein are illustrated for the compound of Example 1:

which is designated 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo-[3.2.1]oct-3-yl}amide, according to the AutoNom software, provided by MDL Information Systems, GmbH (Frankfurt, Germany). The designation (1S,3R,5R) describes the relative orientation of the bonds associated with the bicyclic ring system that are depicted as solid and dashed wedges. The compound is alternatively denoted as N-[(3-endo)-8-[2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl]-3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid. In all of the compounds of the invention depicted herein, unless otherwise specified, the benzoimidazolone-carboxamide is endo to the azabicyclooctane group.

Particular mention may be made of the following compounds:

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo-[3.2.1]oct-3-yl}amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{2-hydroxy-3-[4-(tetrahydrofuran-2-carbonyl)piperazin-1-yl]propyl}-8-azabicyclo-[3.2.1]oct-3-yl)amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[3-(4-acetylpiperazin-1-yl)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{3-[(1,1-dioxotetrahydro-1λ⁶-thiophen-3-yl)methylamino]-2-hydroxypropyl}-8-aza-bicyclo[3.2.1]oct-3-yl)amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(S)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(S)-3-(4-acetylpiperazin-1-yl)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(4-acetylpiperazin-1-yl)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{(S)-2-hydroxy-3-[4-((R)-tetrahydrofuran-2-carbonyl)piperazin-1-yl]propyl}-8-aza-bicyclo[3.2.1]oct-3-yl)amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{(S)-2-hydroxy-3-[4-((S)-tetrahydrofuran-2-carbonyl)piperazin-1-yl]propyl}-8-aza-bicyclo[3.2.1]oct-3-yl)amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(acetylmethylamino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{(R)-2-hydroxy-3-[methyl-(1-methyl-1H-imidazole-4-sulfonyl)amino]propyl}-8-azabicyclo[3.2.1]oct-3-yl)amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(1-methylureido)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{2-hydroxy-3-[methyl-(1-methyl-1H-imidazole-4-sulfonyl)amino]propyl}-8-azabicyclo-[3.2.1]oct-3-yl)amide;

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[2-hydroxy-3-(1-methylureido)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide; and

3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[3-(formylmethylamino)-2-hydroxypropyl]-8-azabicyclo-[3.2.1]oct-3-yl}amide.

Other embodiments of the invention include compounds of formula (I) exemplified herein, such as a compound of formula (I), wherein the compound is 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(acetylmethylamino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide.

As exemplified by particular compounds listed above, the compounds of the invention may contain one or more chiral centers. For example, the carbon atom in formula (I) bearing the substituent —OR² is optionally a chiral center. Accordingly, the invention includes racemic mixtures, pure stereoisomers, and stereoisomer-enriched mixtures of such isomers, unless otherwise indicated. When a particular stereoisomer is shown, it will be understood by those skilled in the art, that minor amounts of other stereoisomers may be present in the compositions of the invention unless otherwise indicated, provided that any utility of the composition as a whole is not eliminated by the presence of such other isomers.

Definitions

When describing the compounds, compositions and methods of the invention, the following terms have the following meanings, unless otherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group which may be linear or branched or combinations thereof. Representative alkyl groups include, by way of example, methyl, ethyl, n-propyl (n-Pr), isopropyl (i-Pr), n-butyl (n-Bu), sec-butyl, isobutyl, tert-butyl, and the like.

The term “halo” means fluoro, chloro, bromo or iodo.

The term “compound” means a compound that was synthetically prepared or produced in any other way, such as by metabolism.

The term “compound of the invention” or “compound of formula I” or “compound of formula I-a” as used herein includes pharmaceutically acceptable salts or solvates or stereoisomers of such compounds unless otherwise indicated.

The term “therapeutically effective amount” means an amount sufficient to effect treatment when administered to a patient in need of treatment.

The term “treatment” as used herein means the treatment of a disease, disorder, or medical condition in a patient, such as a mammal (particularly a human) which includes:

-   -   (a) preventing the disease, disorder, or medical condition from         occurring, i.e., prophylactic treatment of a patient;     -   (b) ameliorating the disease, disorder, or medical condition,         i.e., eliminating or causing regression of the disease,         disorder, or medical condition in a patient;     -   (c) suppressing the disease, disorder, or medical condition,         i.e., slowing or arresting the development of the disease,         disorder, or medical condition in a patient; or     -   (d) alleviating the symptoms of the disease, disorder, or         medical condition in a patient.

The term “pharmaceutically-acceptable salt” means a salt prepared from an acid or base which is acceptable for administration to a patient, such as a mammal. Such salts can be derived from pharmaceutically-acceptable inorganic or organic acids and from pharmaceutically-acceptable bases. Typically, pharmaceutically-acceptable salts of compounds of the present invention are prepared from acids.

Salts derived from pharmaceutically-acceptable acids include, but are not limited to, acetic, adipic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic, xinafoic (1-hydroxy-2-naphthoic acid), naphthalene-1,5-disulfonic acid and the like.

The term “salt thereof” means a compound formed when the hydrogen of an acid is replaced by a cation, such as a metal cation or an organic cation and the like. In the present invention, the cation typically comprises a protonated form of a compound of formula I, i.e. where one or more amino groups having been protonated by an acid. Preferably, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient.

The term “solvate” means a complex or aggregate formed by one or more molecules of a solute, i.e. a compound of the invention or a pharmaceutically-acceptable salt thereof, and one or more molecules of a solvent. Such solvates are typically crystalline solids having a substantially fixed molar ratio of solute and solvent. Representative solvents include by way of example, water, methanol, ethanol, isopropanol, acetic acid, and the like. When the solvent is water, the solvate formed is a hydrate.

It will be appreciated that the term “or a pharmaceutically-acceptable salt or solvate of stereoisomer thereof” is intended to include all permutations of salts, solvates and stereoisomers, such as a solvate of a pharmaceutically-acceptable salt of a stereoisomer of a compound of formula (I).

The term “leaving group” means a functional group or atom which can be displaced by another functional group or atom in a substitution reaction, such as a nucleophilic substitution reaction. By way of example, representative leaving groups include chloro, bromo and iodo groups; sulfonic ester groups, such as mesylate, tosylate, brosylate, nosylate and the like; and acyloxy groups, such as acetoxy, trifluoroacetoxy and the like.

The term “protected derivative thereof” means a derivative of the specified compound in which one or more functional groups of the compound are protected from undesired reactions with a protecting or blocking group. Functional groups which may be protected include, by way of example, carboxylic acid groups, amino groups, hydroxyl groups, thiol groups, carbonyl groups and the like. Representative protecting groups for carboxylic acids include esters (such as a p-methoxybenzyl ester), amides and hydrazides; for amino groups, carbamates (such as tert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters; for thiol groups, thioethers and thioesters; for carbonyl groups, acetals and ketals; and the like. Such protecting groups are well-known to those skilled in the art and are described, for example, in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.

The term “amino-protecting group” means a protecting group suitable for preventing undesired reactions at an amino nitrogen. Representative amino-protecting groups include, but are not limited to, formyl; acyl groups, for example alkanoyl groups, such as acetyl; alkoxycarbonyl groups, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl (Bn), trityl (Tr), and 1,1-di-(4′-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBDMS); and the like.

General Synthetic Procedures

Compounds of the invention can be prepared from readily available starting materials using the following general methods and procedures. Although a particular aspect of the present invention is illustrated in the schemes below, those skilled in the art will recognize that all aspects of the present invention can be prepared using the methods described herein or by using other methods, reagents and starting materials known to those skilled in the art. It will also be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group, as well as suitable conditions for protection and deprotection, are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.

The substituents and variables shown in the following schemes have the definitions provided herein unless otherwise indicated.

In one method of synthesis, compounds of formula (I) can be prepared as illustrated in Scheme A.

An azetidine intermediate (III) is reacted with intermediate (IV), a secondary amine or a derivative of a primary amine, to provide a compound of formula (I). In Scheme A, L′ represents a counterion, such as halide, for example, Cl⁻, Br⁻, or trifluoroacetate. Typically, the azetidine intermediate (III) is dissolved in an inert diluent, such as ethanol, and contacted with between about 1 and about 8 equivalents of the secondary amine (IV), in the presence of a base, such as N,N-diisopropylethylamine. This reaction is typically conducted at a temperature of from about 0° C. to about 100° C. for between about 2 and about 24 hours or until the reaction is substantially complete.

The product of formula (I) is isolated and purified by conventional procedures. For example, the product can be concentrated to dryness under reduced pressure, taken up in an aqueous weak acid solution and purified by HPLC chromatography.

In another method of synthesis, compounds of formula (I) can be prepared as illustrated in Scheme B.

In Scheme B, a different azetidine intermediate (V) is reacted with a benzoimidazolone-carboxamide tropane intermediate (VI) to provide a compound of formula (I). In Scheme B, L′ is a counterion, such as halide, for example, Cl⁻, Br⁻, or trifluoroacetate. In this process, intermediate (V) is dissolved in an inert diluent, such as ethanol, and contacted with between about 1 and about 8 equivalents of the benzoimidazolone-carboxamide tropane (VI). This reaction is typically conducted at a temperature of from about 0° C. to about 100° C. for between about 2 and about 24 hours or until the reaction is substantially complete.

It will be understood that in the process of Scheme B and in other processes described herein using intermediate (VI), intermediate (VI) can be supplied in the form of the free base or in a salt form, with appropriate adjustment of reaction conditions, as necessary, as known to those skilled in the art.

Schemes C and D below are useful for preparing compounds of formula (I) in which the stereochemistry at the center marked by the asterisk is specifically (R) or (S) as well as for preparing non-chiral compounds of formula (I).

In yet another alternate method of synthesis, compounds of formula (I-a), wherein R² is hydrogen, and the carbon atom bonded to —OR² is optionally chiral, can be prepared as illustrated in Scheme C.

A benzoimidazolone-carboxamide tropane intermediate (VI) is reacted with an oxirane-amine intermediate (VII), wherein the * denotes a chiral center, to provide a compound of formula (I-a). As shown in Scheme C, the benzoimidazolone-carboxamide tropane (VI) is contacted with at least one equivalent of an oxirane-amine (VII) in an inert diluent, such as toluene, hexane, or ethanol, to form a compound of formula (I-a). The reaction is typically conducted at a temperature of from about 0° C. to about 100° C. for between about 12 hours and about 24 hours or until the reaction is substantially complete.

In yet another method of synthesis, compounds of formula (I) wherein the carbon atom bonded to —OR² is optionally chiral can be prepared as illustrated in Scheme D.

In Scheme D, an intermediate (VIII), wherein the * denotes a chiral center, is reacted with intermediate (IX), wherein L¹ represents a leaving group, such as chloro, bromo, iodo, or ethoxy, and R⁴ is as defined herein.

Optimal reaction conditions for the reaction of Scheme D may vary depending on the chemical properties of the reagent L¹-R⁴, as is well known to those skilled in the art.

For example, when L¹ is a leaving group, such as chloro, the reaction is typically conducted by contacting intermediate (VIII) with between about 1 and about 4 equivalents of intermediate (IX) in an inert diluent, such as dichloromethane or dimethylformamide, in the presence of an excess of base, for example, between about 3 and about 6 equivalents of base, such as N,N-diisopropylethylamine or 1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU). Suitable inert diluents also include N,N-dimethylformamide, trichloromethane, 1,1,2,2-tetrachloroethane, tetrahydrofuran, and the like. The reaction is typically conducted at a temperature in the range of about −10° C. to about 35° C. for about a quarter hour to about 2 hours, or until the reaction is substantially complete. Exemplary reagents L¹-R⁴ in which L¹ is halo include methanesulfonylchloride and acetylchloride.

When L¹ is leaving group such as ethoxy, for example, when L¹-R⁴ is ethyl formate, the reaction is typically conducted by contacting intermediate (VIII) with between about 1 and about 4 equivalents of intermediate (IX) in an inert diluent, such as dimethylformamide, in the presence of an excess of base, for example, between about 3 and about 6 equivalents of base, such as N,N-diisopropylethylamine. The reaction is typically conducted at a temperature in the range of about 50° C. to about 100° C. for about 22 to about 24 hours, or until the reaction is substantially complete.

Alternatively, intermediate (VIII) can be reacted with a protected isocyanate, such as O═C═NP¹, wherein P¹ is an amino-protecting group, such as trimethylsilyl (TMS), to provide a compound of formula (I) wherein the carbon atom bonded to —OR² is optionally chiral. The reaction is typically conducted by contacting intermediate (VIII) with between about 1 and about 4 equivalents of a protected isocyanate in an inert diluent, such as dimethylformamide, in the presence of an excess of base, for example, between about 3 and about 6 equivalents of base, such as N,N-diisopropyl-ethylamine. The reaction is typically conducted at a temperature in the range of about 0° C. to about 35° C. for about a quarter hour to about 2 hours, or until the reaction is substantially complete. The amino-protecting group can easily be removed as known to those of skill in the art. For example, quenching the reaction mixture with acetic acid and water will hydrolyze the trimethylsilyl (TMS) group to yield a primary urea.

Intermediate compounds of formula (VIII) can be made from readily available compounds. For example, an intermediate compound of formula (VIII) can be prepared from the reaction of a compound of formula (III) and R³NH₂. An alternate way of preparing compounds of formula (III) is also discussed in Scheme G herein.

An azetidine intermediate (III) can be prepared by the procedure illustrated in Scheme E.

In Scheme E, L′ is a counterion, such as halide, for example, Cl⁻ or Br⁻, or trifluoroacetate.

First, an intermediate of formula (VI) is reacted with intermediate (a), wherein L² represents a leaving group, such as bromo, to form an azetidine salt of formula (III-a) in which R² is hydrogen. Intermediate (a) can, for example, be 2-bromomethyloxirane (commonly epibromohydrin). This reaction is typically conducted by contacting intermediate (VI) with between about 2 and about 4 equivalents of the oxirane in a polar diluent, such as ethanol. The reaction is typically conducted at ambient temperature for between about 24 and about 48 hours or until the reaction is substantially complete.

An intermediate of formula (III-b) in which R² is C₁₋₃alkyl, can be prepared by contacting intermediate (III-a) with from slightly less than one equivalent to about one equivalent of a compound of formula L³-R², wherein L³ is a leaving group, such as halo, and R² is as defined herein, in an inert diluent in the presence of between about 1 and about 3 equivalents of a strong base, such as potassium tert-butoxide or sodium hydride. The reaction is typically conducted at ambient temperature for between about a quarter hour to an hour, or until the reaction is substantially complete. Suitable inert diluents include dichloromethane, trichloromethane, 1,1,2,2-tetrachloroethane, and the like.

Similarly, an azetidine intermediate of formula (V) can be prepared by reacting a secondary amine intermediate (IV) with an oxirane intermediate (a), such as epibromohydrin, to provide intermediate (V) in which R² is hydrogen (denoted as (V-a), under the conditions described for Scheme E. An intermediate of formula (V) in which R² is C₁₋₃alkyl (denoted as (V-b)), can be prepared by contacting intermediate (V-a) with a compound of formula L³-R², intermediate (b), as described in Scheme E. A representative method of preparing intermediate (V) is described in Example 5 herein.

An intermediate compound of formula (VII) can be prepared as described in Scheme F.

Intermediate compounds of formula (VII) are typically prepared by reacting intermediate (IV) with intermediate (a), wherein L² is a leaving group, such as chloro, and the * indicates optional R or S chirality, to provide a propanol intermediate (VII′), which is then cyclized to provide a compound of formula (VII).

Typically, an amine compound of formula (IV) is dissolved in an inert diluent, such as ethanol, and contacted with between about 1 and about 8 equivalents of an oxirane intermediate (a), such as epichlorohydrin, to provide a propanol intermediate of formula (VII′). Intermediate (VII′), dissolved in an inert diluent, such as tetrahydrofuran, in the presence of a strong base, such as sodium hydroxide, cyclizes to form intermediate (VII). Each step of this reaction is typically conducted at a temperature of from about 0° C. to about 80° C. for between about 2 and about 24 hours or until the reaction is substantially complete.

Alternatively, a compound of formula (VII′) can be reacted with a compound of formula (VI) to form a compound of formula (I-a). This reaction is typically conducted by contacting a compound of formula (VI) with between about 1 and about 3 equivalents of a compound of formula (VII′) in an inert diluent, such as methanol or ethanol, in the presence of an excess of a base, such as N,N-diisopropyl-ethylamine, to provide a compound of formula (I-a).

An intermediate compound of formula (VIII) can be prepared as shown below in Scheme G.

In Scheme G, a compound of formula (VI) is reacted with intermediate (c), wherein P² is an amino-protecting group, such as Boc, and the * indicates optional R or S chirality, to produce a protected compound (VIII-a) wherein R² is hydrogen. To prepare a compound of formula (VIII-b) in which R² is C₁₋₃alkyl, intermediate (VIII-a) is reacted with a compound of formula L³-R², wherein L³ is a leaving group and R² is as defined, to provide a compound of formula (VIII-b). The amino-protecting group is removed from intermediate (VIII-a) or (VIII-b) to provide a compound of formula (VIII). A representative method of preparing a compound of formula (VIII) using Scheme G, and also a representative method of preparing intermediate (c) are further described in Example 12.

A compound of formula (VI) can be prepared as shown below in Scheme H.

In Scheme H, intermediate (d), a 1,3-dihydrobenzoimidazol-2-one, is dissolved in an inert diluent, such as tetrahydrofuran, in the presence of a strong base, such as sodium hydride, and is reacted with 4-nitrophenyl chloroformate. The mixture is stirred at about 0° C. to about 40° C. for between about 12 and about 24 hours or until the reaction is substantially complete to form an activated ester, intermediate (e) which is reacted, in situ, with a protected amino-tropane, intermediate (f), wherein P³ represents an amino protecting group, such as Boc, in the presence of an inert diluent, such as tetrahydrofuran, at a temperature range of from about 30° C. to about 90° C. for between about 10 and about 24 hours to provide protected intermediate (g). Using conventional methods, the amino-protecting group, P³ is removed from intermediate (g) to provide a benzoimidazolone-carboxamide tropane compound of formula (VI). A representative method of preparing intermediate (VI) is further described in Example 1, steps (a)-(h).

The benzoimidazolone compound of intermediate (d) can be prepared as shown below in Scheme I.

In Scheme I, 2-fluoro-nitrobenzene is reacted with a primary amine, intermediate (h), wherein R¹ is as described herein, to provide intermediate (i), which is reduced to a diaminophenyl, intermediate (j). The diaminophenyl is reacted with carbonyldiimidazole in the presence of an inert diluent, such as tetrahydrofuran, at a temperature range of from about 20° C. to about 40° C. for between about 12 and about 30 hours, to provide a benzoimidazolone compound of intermediate (d). A representative synthesis of a compound of intermediate (d) is described herein in Example 1, steps (a)-(c).

The protected aminotropane, intermediate (f) employed in the reactions described in this application is prepared from readily available starting materials. For example, when the amino-protecting group P³ is Boc, the protected aminotropane can be prepared as shown below in Scheme J, and as further described in steps (d)-(f) of Example 1.

To prepare the protected intermediate (d), 2,5-dimethoxy tetrahydrofuran is contacted with between about 1 and 2 equivalents of benzyl amine and a slight excess, for example about 1.1 equivalents, of 1,3-acetonedicarboxylic acid in an acidic aqueous solution in the presence of a buffering agent such as sodium hydrogen phosphate. The reaction mixture is heated to between about 60° C. and about 100° C. to ensure decarboxylation of any carboxylated intermediates in the product, 8-benzyl-8-azabicyclo-[3.2.1]octan-3-one, commonly N-benzyltropanone.

The N-benzyltropanone intermediate is typically reacted with a slight excess of di-tert-butyl dicarbonate (commonly (Boc)₂O), for example, about 1.1 equivalents, under a hydrogen atmosphere in the presence of a transition metal catalyst to provide 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester. The reaction is typically conducted at ambient temperature for about 12 to about 72 hours. Finally, 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester is contacted with a large excess, for example at least about 25 equivalents, of ammonium formate in an inert diluent, such as methanol, in the presence of a transition metal catalyst to provide the product, intermediate (d), in the endo configuration with high stereospecificity, for example endo to exo ratio of >99:1. The reaction is typically conducted at ambient temperature for about 12 to about 72 hours or until the reaction is substantially complete. It is advantageous to add the ammonium formate reagent in portions. For example, 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester is contacted with an initial portion of ammonium formate of about 15 to about 25 equivalents. After an interval of about 12 to about 36 hours, an additional portion of about 5 to about 10 equivalents of ammonium formate is added. The subsequent addition can be repeated after a similar interval. The product, intermediate (f), can be purified by conventional procedures, such as alkaline extraction.

An oxirane compound of intermediate (c), wherein P² is Boc, can be prepared as shown below in Scheme K.

A benzylamine, intermediate (k), such as N-benzylmethylamine, is contacted with at least one equivalent of a chiral oxirane, intermediate (a), such as epichlorohydrin, in a non-polar diluent, such as hexane or toluene, to form a 2-hydroxypropylamine, intermediate (1). The reaction is typically conducted at room temperature for between about 12 and about 24 hours, or until the reaction is substantially complete. Intermediate (1) is typically reacted with a slight excess of di-tert-butyl dicarbonate (commonly (Boc)₂O), for example, about 1.1 equivalents, under a hydrogen atmosphere in the presence of a transition metal catalyst to provide the Boc protected intermediate (m). The reaction is typically conducted at ambient temperature for between about 8 to about 24 hours. Intermediate (m) is then converted to a cyclized form by dissolving intermediate (m) in an inert diluent, for example, tetrahydrofuran, in the presence of a base, for example, sodium hydroxide, to provide intermediate (c).

The reagents L¹-R⁴, L³-R², HNR³R⁴, and oxirane intermediate (a) are available commercially or are readily prepared by standard procedures from common starting materials.

Further details regarding specific reaction conditions and other procedures for preparing representative compounds of the invention or intermediates thereto are described in the examples below.

Accordingly, in a method aspect, the invention provides a process for preparing a compound of formula (I):

wherein R¹, R², R³, and R⁴ are as defined herein, or a salt or stereoisomer thereof, the process comprising:

(a) reacting a compound of formula (III):

with a compound of the formula (IV):

(b) reacting a compound of formula (V):

with a compound of formula (VI):

(c) reacting a compound of formula (VIII):

with a compound of formula (IX): L¹-R⁴  (IX) wherein L¹ is a leaving group and R⁴ is as defined herein; or

(d) reacting a compound of formula (VIII) with O═C═NP¹, wherein P¹ is an amino-protecting group, and then removing the amino-protecting group P¹;

to provide a compound of formula (I), or a salt or stereoisomer thereof.

The invention also provides a process for preparing a compound of formula (I-a):

wherein R¹, R³, and R⁴ are as defined herein, or a salt or stereoisomer thereof, the process comprising:

reacting a compound of formula (VI):

with a compound of formula (VII):

reacting a compound of formula (VI), with a compound of formula (VII′):

wherein L² is a leaving group;

to provide a compound of formula (I), or a salt or stereoisomer thereof.

In other embodiments, this invention is directed to any of the processes described herein; and to the products prepared by said processes.

The invention also provides a compound of formula (VIII), wherein R¹, R² and R³ are as defined herein for a compound of formula (I), or a salt or stereoisomer or protected derivative thereof.

The invention also provides a compound of formula (I), wherein the compound is prepared by a process comprising:

(a) reacting a compound of formula (III) with a compound of formula (IV);

(b) reacting a compound of formula (V) with a compound of formula (VI);

(c) reacting a compound of formula (VIII) with a compound of formula (IX), wherein L¹ is a leaving group and R⁴ is as defined herein;

(d) reacting a compound of formula (VIII) with O═C═NP¹, wherein P¹ is an amino-protecting group, and then removing the amino-protecting group P¹;

(e) when R² is hydrogen, reacting a compound of formula (VI) with a compound of formula (VII); or

(f) when R² is hydrogen, reacting a compound of formula (VI) with a compound of formula (VII′), wherein L² is a leaving group; to provide a compound of formula (I).

Pharmaceutical Compositions

The benzoimidazolone-carboxamide compounds of the invention are typically administered to a patient in the form of a pharmaceutical composition. Such pharmaceutical compositions may be administered to the patient by any acceptable route of administration including, but not limited to, oral, rectal, vaginal, nasal, inhaled, topical (including transdermal) and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention is directed to a pharmaceutical composition comprising a pharmaceutically-acceptable carrier or excipient and a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. Optionally, such pharmaceutical compositions may contain other therapeutic and/or formulating agents if desired.

The pharmaceutical compositions of the invention typically contain a therapeutically effective amount of a compound of the present invention or a pharmaceutically-acceptable salt thereof. Typically, such pharmaceutical compositions will contain from about 0.1 to about 95% by weight of the active agent; preferably, from about 5 to about 70% by weight; and more preferably from about 10 to about 60% by weight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceutical compositions of the invention. The choice of a particular carrier or excipient, or combinations of carriers or excipients, will depend on the mode of administration being used to treat a particular patient or type of medical condition or disease state. In this regard, the preparation of a suitable pharmaceutical composition for a particular mode of administration is well within the scope of those skilled in the pharmaceutical arts. Additionally, the ingredients for such compositions are commercially available from, for example, Sigma, P.O. Box 14508, St. Louis, Mo. 63178. By way of further illustration, conventional formulation techniques are described in Remington: The Science and Practice of Pharmacy, 20^(th) Edition, Lippincott Williams & White, Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams & White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, the following: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, such as microcrystalline cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical compositions.

The pharmaceutical compositions of the invention are typically prepared by thoroughly and intimately mixing or blending a compound of the invention with a pharmaceutically-acceptable carrier and one or more optional ingredients. If necessary or desired, the resulting uniformly blended mixture can then be shaped or loaded into tablets, capsules, pills and the like using conventional procedures and equipment.

The pharmaceutical compositions of the invention are preferably packaged in a unit dosage form. The term “unit dosage form” means a physically discrete unit suitable for dosing a patient, i.e., each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic effect either alone or in combination with one or more additional units. For example, such unit dosage forms may be capsules, tablets, pills, and the like.

In a preferred embodiment, the pharmaceutical compositions of the invention are suitable for oral administration. Suitable pharmaceutical compositions for oral administration may be in the form of capsules, tablets, pills, lozenges, cachets, dragees, powders, granules; or as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; and the like; each containing a predetermined amount of a compound of the present invention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., as capsules, tablets, pills and the like), the pharmaceutical compositions of the invention will typically comprise a compound of the present invention as the active ingredient and one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate. Optionally or alternatively, such solid dosage forms may also comprise: (1) fillers or extenders, such as starches, microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and/or sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and/or glycerol monostearate; (8) absorbents, such as kaolin and/or bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and/or mixtures thereof; (10) coloring agents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions of the invention. Examples of pharmaceutically-acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Coating agents for tablets, capsules, pills and like, include those used for enteric coatings, such as cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), hydroxypropyl methylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymers, cellulose acetate trimellitate (CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methyl cellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the present invention may also be formulated to provide slow or controlled release of the active ingredient using, by way of example, hydroxypropyl methyl cellulose in varying proportions; or other polymer matrices, liposomes and/or microspheres.

In addition, the pharmaceutical compositions of the present invention may optionally contain opacifying agents and may be formulated so that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way of illustration, pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Such liquid dosage forms typically comprise the active ingredient and an inert diluent, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (such as cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Suspensions, in addition to the active ingredient, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Alternatively, the pharmaceutical compositions of the invention are formulated for administration by inhalation. Suitable pharmaceutical compositions for administration by inhalation will typically be in the form of an aerosol or a powder. Such compositions are generally administered using well-known delivery devices, such as a metered-dose inhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, the pharmaceutical compositions of the invention will typically comprise the active ingredient and a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

Additionally, the pharmaceutical composition may be in the form of a capsule or cartridge (made, for example, from gelatin) comprising a compound of the invention and a powder suitable for use in a powder inhaler. Suitable powder bases include, by way of example, lactose or starch.

The compounds of the invention can also be administered transdermally using known transdermal delivery systems and excipients. For example, a compound of the invention can be admixed with permeation enhancers, such as propylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and the like, and incorporated into a patch or similar delivery system. Additional excipients including gelling agents, emulsifiers and buffers, may be used in such transdermal compositions if desired.

The following formulations illustrate representative pharmaceutical compositions of the present invention:

Formulation Example A

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 50 mg Lactose (spray-dried) 200 mg  Magnesium stearate 10 mg

-   -   Representative Procedure: The ingredients are thoroughly blended         and then loaded into a hard gelatin capsule (260 mg of         composition per capsule).

Formulation Example B

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 20 mg Starch 89 mg Microcrystalline cellulose 89 mg Magnesium stearate  2 mg

-   -   Representative Procedure: The ingredients are thoroughly blended         and then passed through a No. 45 mesh U.S. sieve and loaded into         a hard gelatin capsule (200 mg of composition per capsule).

Formulation Example C

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 10 mg Polyoxyethylene sorbitan monooleate 50 mg Starch powder 250 mg 

-   -   Representative Procedure: The ingredients are thoroughly blended         and then loaded into a gelatin capsule (310 mg of composition         per capsule).

Formulation Example D

Tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention   5 mg Starch  50 mg Microcrystalline cellulose  35 mg Polyvinylpyrrolidone (10 wt. % in water)   4 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc   1 mg

-   -   Representative Procedure: The active ingredient, starch and         cellulose are passed through a No. 45 mesh U.S. sieve and mixed         thoroughly. The solution of polyvinylpyrrolidone is mixed with         the resulting powders, and this mixture is then passed through a         No. 14 mesh U.S. sieve. The granules so produced are dried at         50-60° C. and passed through a No. 18 mesh U.S. sieve. The         sodium carboxymethyl starch, magnesium stearate and talc         (previously passed through a No. 60 mesh U.S. sieve) are then         added to the granules. After mixing, the mixture is compressed         on a tablet machine to afford a tablet weighing 100 mg.

Formulation Example E

Tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 25 mg Microcrystalline cellulose 400 mg  Silicon dioxide fumed 10 mg Stearic acid  5 mg

-   -   Representative Procedure: The ingredients are thoroughly blended         and then compressed to form tablets (440 mg of composition per         tablet).

Formulation Example F

Single-scored tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 15 mg Cornstarch 50 mg Croscarmellose sodium 25 mg Lactose 120 mg  Magnesium stearate  5 mg

-   -   Representative Procedure: The ingredients are thoroughly blended         and compressed to form a single-scored tablet (215 mg of         compositions per tablet).

Formulation Example G

A suspension for oral administration is prepared as follows:

Ingredients Amount Compound of the invention 0.1 g Fumaric acid 0.5 g Sodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 g Granulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k (Vanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilled water q.s. to 100 mL

-   -   Representative Procedure: The ingredients are mixed to form a         suspension containing 10 mg of active ingredient per 10 mL of         suspension.

Formulation Example H

A dry powder for administration by inhalation is prepared as follows:

Ingredients Amount Compound of the invention 1.0 mg Lactose  25 mg

-   -   Representative Procedure: The active ingredient is micronized         and then blended with lactose. This blended mixture is then         loaded into a gelatin inhalation cartridge. The contents of the         cartridge are administered using a powder inhaler.

Formulation Example I

A dry powder for administration by inhalation in a metered dose inhaler is prepared as follows:

-   -   Representative Procedure: A suspension containing 5 wt. % of a         compound of the invention and 0.1 wt. % lecithin is prepared by         dispersing 10 g of active compound as micronized particles with         mean size less than 10 μm in a solution formed from 0.2 g of         lecithin dissolved in 200 mL of demineralized water. The         suspension is spray dried and the resulting material is         micronized to particles having a mean diameter less than 1.5 μm.         The particles are loaded into cartridges with pressurized         1,1,1,2-tetrafluoroethane.

Formulation Example J

An injectable formulation is prepared as follows:

Ingredients Amount Compound of the invention 0.2 g Sodium acetate buffer solution (0.4 M) 40 mL HCl (0.5 N) or NaOH (0.5 N) q.s. to pH 4 Water (distilled, sterile) q.s. to 20 mL

-   -   Representative Procedure: The above ingredients are blended and         the pH is adjusted to 4±0.5 using 0.5 N HCl or 0.5 N NaOH.

Formulation Example K

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the Invention 4.05 mg Microcrystalline cellulose (Avicel PH 103) 259.2 mg  Magnesium stearate 0.75 mg

-   -   Representative Procedure: The ingredients are thoroughly blended         and then loaded into a gelatin capsule (Size #1, White, Opaque)         (264 mg of composition per capsule).

Formulation Example L

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the Invention  8.2 mg Microcrystalline cellulose (Avicel PH 103) 139.05 mg  Magnesium stearate 0.75 mg

-   -   Representative Procedure: The ingredients are thoroughly blended         and then loaded into a gelatin capsule (Size #1, White, Opaque)         (148 mg of composition per capsule).

It will be understood that any form of the compounds of the invention, (i.e. free base, pharmaceutical salt, or solvate) that is suitable for the particular mode of administration, can be used in the pharmaceutical compositions discussed above.

Utility

The benzoimidazolone-carboxamide compounds of the invention are 5-HT₄ receptor agonists and therefore are expected to be useful for treating medical conditions mediated by 5-HT₄ receptors or associated with 5-HT₄ receptor activity, i.e. medical conditions which are ameliorated by treatment with a 5-HT₄ receptor agonist. Such medical conditions include, but are not limited to, irritable bowel syndrome (IBS), chronic constipation, functional dyspepsia, delayed gastric emptying, gastroesophageal reflux disease (GERD), gastroparesis, post-operative ileus, intestinal pseudo-obstruction, and drug-induced delayed transit. In addition, it has been suggested that some 5-HT₄ receptor agonist compounds may be used in the treatment of central nervous system disorders including cognitive disorders, behavioral disorders, mood disorders, and disorders of control of autonomic function.

In particular, the compounds of the invention increase motility of the gastrointestinal (GI) tract and thus are expected to be useful for treating disorders of the GI tract caused by reduced motility in mammals, including humans. Such GI motility disorders include, by way of illustration, chronic constipation, constipation-predominant irritable bowel syndrome (C-IBS), diabetic and idiopathic gastroparesis, and functional dyspepsia.

In one aspect, therefore, the invention provides a method of increasing motility of the gastrointestinal tract in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a compound of the invention.

When used to treat disorders of reduced motility of the GI tract or other conditions mediated by 5-HT₄ receptors, the compounds of the invention will typically be administered orally in a single daily dose or in multiple doses per day, although other forms of administration may be used. The amount of active agent administered per dose or the total amount administered per day will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

Suitable doses for treating disorders of reduced motility of the GI tract or other disorders mediated by 5-HT₄ receptors will range from about 0.0007 to about 20 mg/kg/day of active agent, including from about 0.0007 to about 1 mg/kg/day. For an average 70 kg human, this would amount to from about 0.05 to about 70 mg per day of active agent.

In one aspect of the invention, the compounds of the invention are used to treat chronic constipation. When used to treat chronic constipation, the compounds of the invention will typically be administered orally in a single daily dose or in multiple doses per day. Preferably, the dose for treating chronic constipation will range from about 0.05 to about 70 mg per day.

In another aspect of the invention, the compounds of the invention are used to treat irritable bowel syndrome. When used to treat constipation-predominant irritable bowel syndrome, the compounds of the invention will typically be administered orally in a single daily dose or in multiple doses per day. Preferably, the dose for treating constipation-predominant irritable bowel syndrome will range from about 0.05 to about 70 mg per day.

In another aspect of the invention, the compounds of the invention are used to treat diabetic gastroparesis. When used to treat diabetic gastroparesis, the compounds of the invention will typically be administered orally in a single daily dose or in multiple doses per day. Preferably, the dose for treating diabetic gastroparesis will range from about 0.05 to about 70 mg per day.

In yet another aspect of the invention, the compounds of the invention are used to treat functional dyspepsia. When used to treat functional dyspepsia, the compounds of the invention will typically be administered orally in a single daily dose or in multiple doses per day. Preferably, the dose for treating functional dyspepsia will range from about 0.05 to about 70 mg per day.

The invention also provides a method of treating a mammal having a disease or condition associated with 5-HT₄ receptor activity, the method comprising administering to the mammal a therapeutically effective amount of a compound of the invention or of a pharmaceutical composition comprising a compound of the invention.

Since compounds of the invention are 5-HT₄ receptor agonists, such compounds are also useful as research tools for investigating or studying biological systems or samples having 5-HT₄ receptors, or for discovering new 5-HT₄ receptor agonists. Moreover, since compounds of the invention exhibit binding selectivity for 5-HT₄ receptors as compared with binding to receptors of other 5-HT subtypes, particularly 5-HT₃ receptors, such compounds are particularly useful for studying the effects of selective agonism of 5-HT₄ receptors in a biological system or sample. Any suitable biological system or sample having 5-HT₄ receptors may be employed in such studies which may be conducted either in vitro or in vivo. Representative biological systems or samples suitable for such studies include, but are not limited to, cells, cellular extracts, plasma membranes, tissue samples, mammals (such as mice, rats, guinea pigs, rabbits, dogs, pigs, etc.) and the like.

In this aspect of the invention, a biological system or sample comprising a 5-HT₄ receptor is contacted with a 5-HT₄ receptor-agonizing amount of a compound of the invention. The effects of agonizing the 5-HT₄ receptor are then determined using conventional procedures and equipment, such as radioligand binding assays and functional assays. Such functional assays include ligand-mediated changes in intracellular cyclic adenosine monophosphate (cAMP), ligand-mediated changes in activity of the enzyme adenylyl cyclase (which synthesizes cAMP), ligand-mediated changes in incorporation of analogs of guanosine triphosphate (GTP), such as [³⁵S]GTPγS (guanosine 5′-O-(γ-thio)triphosphate) or GTP-Eu, into isolated membranes via receptor catalyzed exchange of GTP analogs for GDP analogs, ligand-mediated changes in free intracellular calcium ions (measured, for example, with a fluorescence-linked imaging plate reader or FLIPR® from Molecular Devices, Inc.), and measurement of mitogen activated protein kinase (MAPK) activation. A compound of the invention may agonize or increase the activation of 5-HT₄ receptors in any of the functional assays listed above, or assays of a similar nature. A 5-HT₄ receptor-agonizing amount of a compound of the invention will typically range from about 1 nanomolar to about 500 nanomolar.

Additionally, the compounds of the invention can be used as research tools for discovering new 5-HT₄ receptor agonists. In this embodiment, 5-HT₄ receptor binding or functional data for a test compound or a group of test compounds is compared to the 5-HT₄ receptor binding or functional data for a compound of the invention to identify test compounds that have superior binding or functional activity, if any. This aspect of the invention includes, as separate embodiments, both the generation of comparison data (using the appropriate assays) and the analysis of the test data to identify test compounds of interest.

Accordingly, in another of its method aspects, this invention relates to a method of evaluating a test compound in a biological assay, the method comprising: (a) conducting a biological assay with a test compound to provide a first assay value; (b) conducting the biological assay with a compound of formula I to provide a second assay value; wherein step (a) is conducted either before, after or concurrently with step (b); and (c) comparing the first assay value from step (a) with the second assay value from step (b).

Among other properties, compounds of the invention have been found to be potent agonists of the 5-HT₄ receptor and to exhibit substantial selectivity for the 5-HT₄ receptor subtype over the 5-HT₃ receptor subtype in radioligand binding assays. Further, compounds of the invention have demonstrated superior pharmacokinetic properties in a rat model. Compounds of the invention are thus expected to demonstrate good bioavailablity upon oral administration. In addition, representative compounds have been shown not to exhibit an unacceptable level of inhibition of the potassium ion current in an in vitro voltage-clamp model using isolated whole cells expressing the hERG cardiac potassium channel. The voltage-clamp assay is an accepted pre-clinical method of assessing the potential for pharmaceutical agents to change the pattern of cardiac repolarization, specifically to cause, so-called QT prolongation, which has been associated with cardiac arrhythmia. (Cavero et al., Opinion on Pharmacotherapy, 2000, 1, 947-73, Fermini et al., Nature Reviews Drug Discovery, 2003, 2, 439-447) Accordingly, pharmaceutical compositions comprising compounds of the invention are expected to have an acceptable cardiac profile.

These properties, as well as the utility of the compounds of the invention, can be demonstrated using various in vitro and in vivo assays well-known to those skilled in the art. Representative assays are described in further detail in the following examples.

EXAMPLES

The following synthetic and biological examples are offered to illustrate the invention, and are not to be construed in any way as limiting the scope of the invention. In the examples below, the following abbreviations have the following meanings unless otherwise indicated. Abbreviations not defined below have their generally accepted meanings.

-   -   Boc=tert-butoxycarbonyl     -   (Boc)₂O=di-tert-butyl dicarbonate     -   DCM=dichloromethane     -   DMF=N,N-dimethylformamide     -   DMSO=dimethyl sulfoxide     -   EtOAc=ethyl acetate     -   LCMS=liquid chromatography mass spectrometry     -   mCPBA=m-chloroperbenzoic acid     -   MeCN=acetonitrile     -   MTBE=tert-butyl methyl ether     -   PyBop=benzotriazol-1-yloxytripyrrolidino-phosphonium         hexafluorophosphate     -   R_(f)=retention factor     -   RT=room temperature     -   TFA=trifluoroacetic acid     -   THF=tetrahydrofuran

Reagents (including secondary amines) and solvents were purchased from commercial suppliers (Aldrich, Fluka, Sigma, etc.), and used without further purification. Reactions were run under nitrogen atmosphere, unless noted otherwise. Progress of reaction mixtures was monitored by thin layer chromatography (TLC), analytical high performance liquid chromatography (anal. HPLC), and mass spectrometry, the details of which are given below and separately in specific examples of reactions. Reaction mixtures were worked up as described specifically in each reaction; commonly they were purified by extraction and other purification methods such as temperature-, and solvent-dependent crystallization, and precipitation. In addition, reaction mixtures were routinely purified by preparative HPLC: a general protocol is described below. Characterization of reaction products was routinely carried out by mass and ¹H-NMR spectrometry. For NMR measurement, samples were dissolved in deuterated solvent (CD₃OD, CDCl₃, or DMSO-d₆), and ¹H-NMR spectra were acquired with a Varian Gemini 2000 instrument (300 MHz) under standard observation conditions. Mass spectrometric identification of compounds was performed by an electrospray ionization method (ESMS) with an Applied Biosystems (Foster City, Calif.) model API 150 EX instrument or an Agilent (Palo Alto, Calif.) model 1100 LC/MSD instrument.

A general protocol for analytical HPLC: Each of crude compounds was dissolved in 50% MeCN/H₂O (with 0.1% TFA) at 0.5-1.0 mg/mL concentration, and was analyzed by using anal. HPLC: 1) reverse-phased anal. Column: Zorbax Bonus-RP (3.5 μm of particle size, 2.1×50 mm); 2) flow rate: 0.5 mL/min; 3) 5% MeCN/H₂O containing 0.1% TFA (isocratic; 0-0.5 min); 5% MeCN/H₂O containing 0.1% TFA to 75% MeCN/H₂O containing 0.1% TFA (linear gradient; 0.5-4 min); 4) detection: 214, 254, and 280 nm. Other conditions used are indicated whenever necessary.

A general protocolfor preparative HPLC purification: Crude compounds were dissolved in 50% acetic acid in water at 50-100 mg/mL concentration, filtered, and fractionated using preparative HPLC: 1) column; YMC Pack-Pro C18 (50a×20 mm; ID=5 μm); 2) linear gradient: 10% A/90% B to 50% A/50% B over 30 min; 3) flow rate: 40 mL/min; 4) detection: 214 nm.

Preparation of Secondary Amines

Preparation of various secondary amines used as intermediates in the synthesis of a compound of formula (I) are described below.

The N-sulfonyl derivatives of piperazine were prepared from N-Boc piperazine by reacting with respective sulfonyl chloride (iPr₂NEt, CH₂Cl₂, 0° C.), and deprotecting the N-Boc group (CF₃CO₂H, CH₂Cl₂). 1-Methanesulfonylpiperazine: ¹H-NMR (CDCl₃; neutral): δ (ppm) 3.1 (t, 4H), 2.9 (t, 4H), 2.7 (s, 3H). Methanesulfonylpiperazine was also prepared by reacting methanesulfonyl chloride with excess piperazine (>2 equivalents) in water.

The racemic or single chiral isomer forms of 3-acetylaminopyrrolidine were prepared by treating N¹-Boc-3-aminopyrrolidine (racemate, 3R, or 3S) with acetyl chloride (iPr₂NEt, CH₂Cl₂, 0° C.), and deprotecting the N-Boc group (CF₃CO₂H, CH₂Cl₂). 3-(Acetamido)pyrrolidine: ¹H-NMR (DMSO-d₆; TFA salt): δ (ppm) 4.2 (quin, 1H), 3.3-3.1 (m, 3H), 2.9 (m, 1H), 2.0 (m, 1H), 1.8 (br s, 4H).

3-((R)-2-Hydroxypropionamido)pyrrolidine was prepared after amidation of N¹-Boc-3-aminopyrrolidine (L-lactic acid, PyBOP, DMF, RT), and deprotection of N-Boc group (CF₃CO₂H, CH₂Cl₂). (m/z): [M+H]⁺ calcd for C₇H₁₄N₂O₂, 159.11; found, 159.0. ¹H-NMR (CD₃OD; TFA salt): δ (ppm) 4.4 (quin, 1H), 4.1 (q, 1H), 3.5-3.4 (m, 2H), 3.3-3.2 (m, 2H), 2.3 (m, 1H), 2.0 (m, 1H), 1.3 (d, 3H).

The N³-alkanesulfonyl derivatives of (3R)-aminopyrrolidine were obtained by treating N¹-Boc-(3R)-aminopyrrolidine with propionylsulfonyl chloride or cyclohexylmethylsulfonyl chloride (i-Pr₂NEt, CH₂Cl₂, 0° C.), and deprotecting N-Boc group (CF₃CO₂H, CH₂Cl₂).

Derivatives of tetrahydro-3-thiophenamine-1,1-dioxide were prepared following the protocol of Loev, B. J. Org. Chem. 1961, 26, 4394-9 by reacting 3-sulfolene with a requisite primary amine in methanol (cat. KOH, RT). N-Methyl-3-tetrahydrothiophene-amine-1,1-dioxide (TFA salt): ¹H-NMR (DMSO-d₆): δ (ppm) 9.4 (br s, 2H), 4.0-3.8 (quin, 1H), 3.6-3.5 (dd, 1H), 3.4-3.3 (m, 1H), 3.2-3.1 (m, 2H), 2.5 (s, 3H), 2.4 (m, 1H), 2.1 (m, 1H).

(S)-1,1-Dioxo-tetrahydro-1λ⁶-thiophen-3-ylamine was prepared as follows:

1) N-Boc protection of (S)-3-tetrahydrothiophenamine (Dehmlow, E. V.; Westerheide, R. Synthesis 1992, 10, 947-9) by treating with (Boc)₂O in methanol at room temperature for about 12 h; 2) oxidation by treating with mCPBA in dichloromethane to N-Boc protected (S)-1,1-dioxo-tetrahydro-1λ⁶-thiophen-3-ylamine at 0° C. for about 5 h (washed multiple times with a saturated sodium sulfite solution to quench trace peroxides); and 3) N-Boc deprotection of the sulfone derivative with TFA in dichloro-methane at room temperature for 1 h to the free amine which was isolated as a TFA salt. (R)-1,1-dioxo-tetrahydro-1λ⁶-thiophen-3-ylamine was prepared using the same method, but replacing the (S)-3-tetrahydrothiophenamine with (R)-3-tetrahydrothiophenamine.

N-Methyl-tetrahydro-2H-thiopyran-4-amine-1,1-dioxide was prepared from tetrahydro-4H-thiopyran-4-one: i) MeNH₂, NaBH₄; ii) (Boc)₂O, MeOH; iii) mCPBA, CH₂Cl₂, 0° C. (washed multiple times with a saturated sodium sulfite solution to quench trace peroxides); iv) CF₃CO₂H, CH₂Cl₂. (m/z): [M+H]⁺ calcd for C₆H₁₃NO₂S 164.07; found, 164.9. ¹H-NMR (CD₃OD; TFA salt): δ (ppm) 3.4-3.1 (m, 5H), 2.7 (s, 3H), 2.4 (br d, 2H), 2.1 (br m, 2H).

Example 1 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo-[3.2.1]oct-3-yl}amide (by Scheme A)

a. Preparation of N-isopropyl-N-(2-nitrophenyl)amine

To a cold solution of 2-fluoro-nitrobenzene (31.8 g, 0.225 mol) in ethanol (300 mL) cooled in an ice bath was added isopropylamine (54.0 mL, 0.634 mol), followed by the addition of a solution of potassium carbonate (31.1 g, 0.225 mol) in water (120 mL). The mixture was stirred at 0° C. for 1 h, then refluxed for 6 h. The reaction was terminated by cooling the mixture to ambient temperature, and evaporating it under reduced pressure yielding an orange residue. The residue was partitioned between ethyl ether (800 mL) and a brine solution (300 mL). The organic layer was dried and filtered, to provide the title intermediate (39 g) as an orange liquid. ¹H-NMR (CDCl₃, 300 MHz): δ (ppm) 8.06 (d, 1H), 7.30 (t, 1H), 6.74 (d, 1H), 6.48 (t, 1H), 3.73 (hept, 1H), 1.20 (d, 6H).

b. Preparation of N-(2-aminophenyl)-N-isopropylamine

To a mixture of ethanol (600 mL) and 2 M sodium hydroxide solution (320 mL) cooled in an ice bath was added Zn dust (59.5 g) slowly. While stirring the Zn slurry, N-isopropyl-N-(2-nitrophenyl)amine (41 g, 0.228 mol) dissolved in ethanol (50 mL) was added. The mixture was stirred at 0° C. for 30 min, then heated to 85° C. The mixture was stirred at 85° C. for about 12 h until the refluxing solution of the mixture became a colorless solution. The mixture was then cooled to 0° C. and filtered. The collected solid was rinsed with ethyl acetate (200 mL). The filtrate and rinsed solution were combined, and evaporated in vacuo to remove excess volatile solvents. During the concentration, the mixture became pale brown/yellow. The aqueous concentrate was extracted with ethyl acetate (800 mL). The organic solution was concentrated to dryness, to provide the title intermediate (33 g) as a brown-pink oil which was used in the next step without further treatment. ¹H-NMR (CDCl₃, 300 MHz): δ (ppm) 6.73-6.5 (m, 4H), 3.58-3.55 (hept, 1H), 1.2 (d, 6H).

c. Preparation of 1-isopropyl-1,3-dihydro-2H-benzimidazol-2-one

To a solution of the product of step (b), N-(2-aminophenyl)-N-isopropylamine (34 g, 0.226 mol), in tetrahydrofuran (500 mL) was added carbonyldiimidazole (36.7 g, 0.226 mol) as a solid. The mixture was stirred under an atmosphere of nitrogen gas at ambient temperature for about 24 h. The mixture was concentrated in vacuo, and a resulting dark brown residue was distributed between ethyl acetate (700 mL) and brine solution (300 mL). The organic layer was then washed with 1 M phosphoric acid multiple times (˜3×300 mL) until the color of the organic layer turned from dark brown to pale yellow. The organic solution was evaporated to dryness to provide the title intermediate (34 g) as a pale yellow oil which solidified slowly on standing. The purity of the material was assessed by ¹H-NMR which indicated no detectable impurity: ¹H-NMR (CD₃OD, 300 MHz): δ (ppm) 7.2 (m, 1H), 7.0 (m, 3H), 4.6 (hept, 1H), 1.46 (d, 6H). (m/z): [M+H]⁺ calcd for C₁₀H₁₂N₂O 177.09; found, 177.2.

Anal. HPLC: retention time=2.7 min (99% purity): 1) column: Zorbax, Bonus-RP, 3.5 μm of particle size, 2.1×50 mm; 2) flow rate: 0.5 mL/min; 3) isocratic condition (10% solvent B/90% solvent A) for 0 to 0.5 min; then linear gradient to 50% solvent B/50% solvent A over 5 min (solvent A=98% water/2% MeCN/0.1% TFA; solvent B=90% MeCN/10% water/0.1% TFA). TLC analysis (silica gel plate): R_(f)=0.5 (CH₂Cl₂). (m/z): [M+H]⁺ calcd for C₁₀H₁₂N₂O 177.09; found 177.3.

d. Preparation of 8-benzyl-8-azabicyclo[3.2.1]octan-3-one

Concentrated hydrochloric acid (30 mL) was added to a heterogeneous solution of 2,5-dimethoxy tetrahydrofuran (82.2 g, 0.622 mol) in water (170 mL) while stirring. In a separate flask cooled to 0° C. (ice bath), concentrated hydrochloric acid (92 mL) was added slowly to a solution of benzyl amine (100 g, 0.933 mol) in water (350 mL). The 2,5-dimethoxytetrahydrofuran solution was stirred for approximately 20 min, diluted with water (250 mL), and then the benzyl amine solution was added, followed by the addition of a solution of 1,3-acetonedicarboxylic acid (100 g, 0.684 mol) in water (400 mL) and then the addition of sodium hydrogen phosphate (44 g, 0.31 mol) in water (200 mL). The pH was adjusted from pH 1 to pH ˜4.5 using 40% NaOH. The resulting solution was stirred overnight. The solution was then acidified to pH 3 from pH 7.5 using 50% hydrochloric acid, heated to 85° C. and stirred for 2 hours. The solution was cooled to room temperature, basified to pH 12 using 40% NaOH, and extracted with DCM (3×500 mL). The combined organic layers were washed with brine, dried, filtered and concentrated under reduced pressure to produce the crude title intermediate as a viscous brown oil (52 g).

To a solution of the crude intermediate in methanol (1000 mL) was added di-tert-butyl dicarbonate (74.6 g, 0.342 mol) at 0° C. The solution was allowed to warm to room temperature and stirred overnight. The methanol was removed under reduced pressure and the resulting oil was dissolved in dichloromethane (1000 mL). The intermediate was extracted into 1 M H₃PO₄ (1000 mL) and washed with dichloromethane (3×250 mL) The aqueous layer was basified to pH 12 using aqueous NaOH, and extracted with dichloromethane (3×500 mL). The combined organic layers were dried, filtered and concentrated under reduced pressure to provide the title intermediate as a viscous, light brown oil. ¹H-NMR (CDCl₃) δ (ppm) 7.5-7.2 (m, 5H, C₆H₅), 3.7 (s, 2H, CH₂Ph), 3.45 (broad s, 2H, CH—NBn), 2.7-2.6 (dd, 2H, CH₂CO), 2.2-2.1 (dd, 2H, CH₂CO), 2.1-2.0 (m, 2H, CH₂CH₂), 1.6 (m, 2H, CH₂CH₂). (m/z): [M+H]⁺ calcd for C₁₄H₁₇NO 216.14; found, 216.0.

e. Preparation of 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester

To a solution of 8-benzyl-8-azabicyclo[3.2.1]octan-3-one (75 g, 0.348 mol) in EtOAc (300 mL) was added a solution of di-tert-butyl dicarbonate (83.6 g, 0.383 mol, 1.1 eq) in EtOAc (300 mL). The resulting solution and rinse (100 mL EtOAc) was added to a 1 L Parr hydrogenation vessel containing 23 g of palladium hydroxide (20 wt. % Pd, dry basis, on carbon, ˜50% wet with water; e.g. Pearlman's catalyst) under a stream of nitrogen. The reaction vessel was degassed (alternating vacuum and N₂ five times) and pressurized to 60 psi of H₂ gas. The reaction solution was agitated for two days and recharged with H₂ as needed to keep the H₂ pressure at 60 psi until the reaction was complete as monitored by silica thin layer chromatography. The black solution was then filtered through a pad of Celite® and concentrated under reduced pressure to provide the title intermediate as a viscous, yellow to orange oil which was used in the next step without further treatment. ¹H NMR (CDCl₃) δ(ppm) 4.5 (broad, 2H, CH—NBoc), 2.7 (broad, 2H, CH₂CO), 2.4-2.3 (dd, 2H, CH₂CH₂), 2.1 (broad m, 2H, CH₂CO), 1.7-1.6 (dd, 2H, CH₂CH₂), 1.5 (s, 9H, (CH₃)₃COCON)).

f. Preparation of (1S,3R,5R)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester

To a solution of the product of the previous step (75.4 g, 0.335 mol) in methanol (1 L) was added ammonium formate (422.5 g, 6.7 mol), water (115 mL) and 65 g of palladium on activated carbon (10% on dry basis, ˜50% wet with water; Degussa type E101NE/W) under a stream of N₂ while stirring via mechanical stirrer. After 24 and 48 hours, additional portions of ammonium formate (132 g, 2.1 mol) were added. Once reaction progression ceased, as monitored by anal. HPLC, Celite® (>500 g) was added and the resulting thick suspension was filtered and then the collected solid was rinsed with methanol (˜500 mL). The filtrates were combined and concentrated under reduced pressure. The resulting cloudy, biphasic solution was then diluted with 1M phosphoric acid to a final volume of ˜1.5 to 2.0 L at pH 2 and washed with dichloromethane (3×700 mL). The aqueous layer was basified to pH 12 using 40% aq. NaOH, and extracted with dichloromethane (3×700 mL). The combined organic layers were dried, filtered, and concentrated by rotary evaporation, then high-vacuum to provide the title intermediate (52 g), commonly N-Boc-endo-3-aminotropane, as a white to pale yellow solid. The isomer ratio of endo to exo amine of the product was >99:1 based on ¹H-NMR analysis (>96% purity by analytical HPLC). ¹H NMR (CDCl₃) δ (ppm) 4.2-4.0 (broad d, 2H, CHNBoc), 3.25 (t, 1H, CHNH₂), 2.1-2.05 (m, 4H), 1.9 (m, 2H), 1.4 (s, 9H, (CH₃)₃OCON), 1.2-1.1 (broad, 2H). (m/z): [M+H]⁺ calcd for C₁₂H₂₂N₂O₂ 227.18; found, 227.2. Analytical HPLC (isocratic method; 2:98 (A:B) to 90:10 (A:B) over 5 min): retention time=3.68 min.

g. Preparation of (1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester

To a cold suspension of sodium hydride (9.25 g; 231.4 mmol; 60% dispersion in mineral oil) in dry THF (1000 L) in an ice bath was added 1-isopropyl-1,3-dihydro-2H-benzimidazol-2-one (27.2 g, 154.2 mmol) in THF (50 mL) under nitrogen atmosphere. The mixture was stirred at ˜0-5° C. for 30 min, then 4-nitrophenyl chloroformate (34.2 g, 170 mmol) in THF (50 mL) was added. The mixture was stirred overnight while allowing the mixture to gradually warm to ambient temperature. To the activated ester formed was then added (1S,3R,5R)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (36.7 g, 162 mmol) in THF (50 mL). The mixture was stirred at ambient temperature for about 12 h, and at about 75° C. for about 3° h, at which time an LCMS of the reaction sample indicated completion of the coupling reaction. The mixture was concentrated in vacuo, dissolved in dichloromethane (1 L), and washed with first 1M H₃PO₄, and then saturated NaHCO₃ solution. After drying, the organic solution was evaporated to provide the title intermediate as a pale yellow residue that was used in the next step without further treatment.

h. Preparation of N-[(1S,3R,5R)-3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid (8-azabicyclo[3.2.1]oct-3-yl)amide

To a cold solution of (1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzo-imidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester in 200 mL of dichloromethane in an ice bath was added trifluoroacetic acid (200 mL). The mixture was stirred for about 30 min at ˜5° C., and at room temperature for about 1 h. After evaporation of the mixture, ethyl ether (˜500 mL) was added to the oily residue, causing solidification of the residue. The precipitate was collected, rinsed with copious amounts of ethyl ether, and dried in vacuo, to provide the title intermediate (47 g) as a TFA salt. The title intermediate is also commonly referred to as endo-N-(8-azabicyclo[3.2.1]oct-3-yl)-3-isopropyl-2-oxo-2,3-dihydrobenzimidazole-1-carboxamide.

i. Preparation of 3-hydroxy-3′-{[3-isopropyl-2-oxo-2,3-dihydrobenzimidazolyl-carbonyl]amino}spiro[azetidine-1.8′-(1S,3R,5R)-8-aza-bicyclo[3.2.1]octane]

To a solution of N-[(1S,3R,5R)-3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid (8-azabicyclo[3.2.1]oct-3-yl)amide (0.884 g, 2 mmol; TFA salt) in ethanol (10 mL) was added first N,N-diisopropylethylamine (0.348 mL, 2 mmol) followed by epibromohydrin (0.274 g, 4 mmol). The mixture was stirred at ambient temperatures overnight and the spiro product was precipitated, collected by filtration and rinsed with cold ethanol, and used in the next step without further treatment.

j. Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo-[3.2.1]oct-3-yl}amide

To a solution of 3-hydroxy-3′-{[3-isopropyl-2-oxo-2,3-dihydrobenzimidazolyl-carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-aza-bicyclo[3.2.1]octane] (0.135 g, 0.35 mmol) in ethanol (10 mL) was added N,N-diisopropylethylamine (0.24 mL, 1.4 mmol) and N-methylsulfonylpiperazine/TFA salt (0.195 g, 0.7 mmol). The mixture was shaken at 80° C. for 12 h, and concentrated in vacuo, yielding a pale yellow oily residue. The crude product was dissolved in 50% aqueous acetic acid, then purified by preparative high performance liquid chromatography to provide the title compound (30.1 mg) as a TFA salt. (m/z): [M+H]⁺ calcd for C₂₆H₄₀N₆O₅S 549.28; found 549.2. Retention time (anal. HPLC: 10-40% MeCN/H₂O over 6 min)=2.13 min.

Example 2 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{2-hydroxy-3-[4-(tetrahydrofuran-2-carbonyl)piperazin-1-yl]propyl}-8-azabicyclo-[3.2.1]oct-3-yl)amide

Using the processes described in Example 1, except in step (j) replacing N-methylsulfonylpiperazine with N-(tetrahydro-2-furoyl)piperazine, the title compound was prepared (23.7 mg) as a TFA salt. (m/z): [M+H]⁺ calcd for C₃₀H₄₄N₆O₅) 569.34; found 569.4. Retention time (anal. HPLC: 10-40% MeCN/H₂O over 6 min)=2.19 min.

Example 3 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[3-(4-acetylpiperazin-1-yl)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

Using the processes described in Example 1, except in step (j) replacing N-methylsulfonylpiperazine with N-acetylpiperazine, the title compound was prepared (23.1 mg) as a TFA salt. (m/z): [M+H]⁺ calcd for C₂₇H₄₀N₆O₄ 513.31; found 513.2. Retention time (anal. HPLC: 10-40% MeCN/H₂O over 6 min)=2.12 min.

Example 4 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{3-[(1,1-dioxotetrahydro-1λ⁶-thiophen-3-yl)methylamino]-2-hydroxypropyl}-8-azabicyclo[3.2.1]oct-3-yl)amide

Using the processes described in Example 1, except in step (j) replacing N-methylsulfonylpiperazine with N-methyltetrahydro-3-thiophenamine-1,1-dioxide, the title compound was prepared (16.3 mg) as a TFA salt. (m/z): [M+H]⁺ calcd for C₂₆H₃₉N₅O₅S 534.27; found 534.2. Retention time (anal. HPLC: 10-40% MeCN/H₂O over 6 min)=2.18 min.

Example 5 Alternative synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo-[3.2.1]oct-3-yl}amide (by Scheme B)

The title compound was prepared by reacting N-methylsulfonylpiperazine/TFA salt (0.6 g, 2.16 mmol) with epibromohydrin (0.591 g, 4.31 mmol) in ethanol (9 mL) containing N,N-diisopropylethylamine (0.375 mL, 2.16 mmol) at ambient temperatures for 12 h. The precipitate, 2-hydroxy-7-methylsulfonyl-7-aza-4-azoniaspiro[3.5]nonane, was then reacted with endo-N-(8-azabicyclo[3.2.1]oct-3-yl)-3-isopropyl-2-oxo-2,3-dihydrobenzimidazole-1-carboxamide, (prepared as described in Example 1, step (h)), in ethanol at about 80° C. for about 12 h, to provide the title compound as a TFA salt. LCMS and anal. HPLC analysis of the product indicated that the compound prepared by this process was identical to the compound prepared in Example 1 by Scheme A.

Example 6 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(S)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-amide (by Scheme C)

a. Preparation of (S)-1-chloro-3-(4-methylsulfonyl-1-piperazinyl)-2-propanol

(S)-Epichlorohydrin (48.0 mL, 0.612 mol) was added to a stirred solution of piperazine N-methylsulfonamide (87.3 g, 0.532 mol) in ethanol (1.33 L) at room temperature. The reaction mixture was stirred for 18 h and the white solid precipitate which formed was collected by filtration and washed with ethanol to provide the title intermediate (107.7 g) as a white solid which was used without further purification. (m/z): [M+H]⁺ calcd for C₈H₁₇ClN₂O₃S, 257.07; found, 257.2. ¹H-NMR (DMSO): δ(ppm) 5.09 (d, 1H), 3.84 (m, 1H), 3.65 (dd, 1H), 3.55 (dd, 1H), 3.09 (m, 4H), 2.37 (dd, 1H), 2.86 (s, 3H), 2.50-2.58 (m, 4H), 2.45 (dd, 1H).

b. Preparation of (S)-1-methylsulfonyl-4-(oxiranylmethyl)-piperazine

Sodium hydroxide (22.15 g, 0.534 mol) was added to a vigorously stirred solution of the product of the previous step (118.13 g, 0.461 mol) in 80% THF in water (1500 mL) at 0° C. The reaction mixture was stirred for 90 min and the layers were separated. The organic layer was concentrated under vacuum, diluted with dichloromethane (1500 mL), and washed with a mixture of the previously separated aqueous layer and 1M NaOH (500 mL). The organic layer was further washed with 1M NaOH (500 mL) and brine (500 mL), dried, filtered and concentrated under vacuum to provide a white crystalline solid (90.8 g). The crystalline solid was recrystallised from a hot 1:1 mixture of EtOAc and hexane (800 mL) to yield 43.33 g of the title intermediate. (m/z): [M+H]⁺ calcd for C₈H₁₆N₂O₃S 221.10; found 221.3. ¹H-NMR (DMSO-d₆): δ(ppm) 3.11 (m, 4H), 3.02 (m, 1H), 2.87 (s, 3H), 2.69-2.75 (m, 2H), 2.45-2.60 (m, 5H), 2.22 (dd, 1H).

c. Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(S)-2-hydroxy-3-(4-methanesulfonypiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-amide

To a solution of endo-N-(8-azabicyclo[3.2.1]oct-3-yl)-3-isopropyl-2-oxo-2,3-dihydrobenzimidazole-1-carboxamide, (prepared as described in Example 1, step (h)), (0.3 g, 0.914 mmol) in toluene (3 mL) was added (S)-1-methylsulfonyl-4-(oxiranylmethyl)-piperazine (0.201 g, 0.913 mmol). The mixture was stirred at 100° C. for 18 h, and concentrated in vacuo, yielding an oily residue. The residue was dissolved in 50% aqueous acetic acid, and purified by preparative HPLC, to provide the title compound (0.135 g) as a TFA salt. (m/z): [M+H]⁺ calcd for C₂₆H₄₀N₆O₅S 549.28; found 549.2. Retention time (anal. HPLC: 10-40% MeCN/H₂O over 6 min)=2.13 min.

Example 7 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-amide

Using the processes described in Example 6, except replacing (S)-1-methyl-sulfonyl-4-(oxiranylmethyl)-piperazine with (R)-1-methylsulfonyl-4-(oxiranylmethyl)-piperazine, the title compound was prepared (0.110 g) as a TFA salt. The LCMS and analytical HPLC profiles of (S)- and (R)-isomer for the compounds of Example 6 and 7 were identical.

Example 8 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(S)-3-(4-acetylpiperazin-1-yl)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide (by Scheme D)

To a solution of endo-N-(8-azabicyclo[3.2.1]oct-3-yl)-3-isopropyl-2-oxo-2,3-dihydrobenzimidazole-1-carboxamide, (prepared as described in Example 1, step (h)) (0.658 g, 2.0 mmol) in ethanol (10 mL) was added (S)-1-acetyl-4-(oxiranylmethyl)-piperazine (0.407 g, 2.21 mmol). The mixture was stirred at 80° C. for 18 h, and concentrated in vacuo, yielding an oily residue. It was dissolved in 50% aqueous acetic acid, and purified by preparative HPLC, to provide the title compound (0.675 g) as a TFA salt. (m/z): [M+H]⁺ calcd for C₂₇H₄₀N₆O₄ 513.31; found 513.2. Retention time (anal. HPLC: 10-40% MeCN/H₂O over 6 min)=2.12 min.

Example 9 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(4-acetylpiperazin-1-yl)-2-hydroxypropyl]-8-azabicyclo [3.2.1]oct-3-yl}amide

Using the processes described in Example 8, except replacing (S)-1-acetyl-4-(oxiranylmethyl)-piperazine with (R)-1-acetyl-4-(oxiranylmethyl)-piperazine, the title compound was prepared (0.604 g) as a TFA salt. The LCMS and analytical HPLC profiles of the (S)- and (R)-isomers of Examples 8 and 9 were identical.

Example 10 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{(S)-2-hydroxy-3-[4-((R)-tetrahydrofuran-2-carbonyl)piperazin-1-yl]propyl}-8-aza-bicyclo[3.2.1]oct-3-yl)amide

a. Preparation of 1-[(R)-tetrahydro-2-furoyl]-4-Boc-piperazine

To a solution of (R)-tetrahydro-2-furoic acid (5 g, 43.06 mmol) in toluene (75 mL) was added thionyl chloride (7.68 g, 64.6 mmol). The mixture was stirred at 90° C. for 2 h, then slowly cooled to 0° C. To a separate flask was charged a solution of N-Boc piperazine (8.02 g, 43.06 mmol) in toluene (40 mL), and aqueous sodium hydroxide (10.3 g, 258 mmol) in water (80 mL). This biphasic solution was cooled in an ice bath, followed by the addition of the cold acid chloride prepared above. The mixture was stirred vigorously for 2 h. The organic phase was collected, and washed with 1M H₃PO₄, dried and evaporated, to provide the title intermediate (9.87 g).

b. Preparation of 1-[(R)-tetrahydro-2-furoyl]piperazine

1-[(R)-Tetrahydro-2-furoyl]-4-Boc-piperazine was dissolved in 100 mL of dichloromethane, cooled in an ice bath, and trifluoroacetic acid (50 mL) was added. The mixture was stirred for 2 h, then concentrated, to provide the title intermediate as a TFA salt. The TFA salt form was converted to a free base by an extraction procedure using dichloromethane and 1M sodium hydroxide solution.

c. Preparation of (S)-1-chloro-3-(4-[(R)-tetrahydro-2-furoyl]-1-piperazinyl)-2-propanol

To a solution of 1-[(R)-tetrahydro-2-furoyl]piperazine (1.89 g, 10.26 mmol) in ethanol (25 mL) was added (S)-epichlorohydrin (1.09 g, 11.8 mmol). The mixture was stirred at room temperature overnight, then concentrated in vacuo, yielding a pale yellow oil. The oil was dissolved in dichloromethane (100 mL), and washed with 1M H₃PO₄, dried and evaporated, to provide the title intermediate.

d. Preparation of 1-[(R)-tetrahydro-2-furoyl]-4-((S)-oxiranylmethyl)-piperazine

To a cold solution of (S)-1-chloro-3-(4-[(R)-tetrahydro-2-furoyl]-1-piperazinyl)-2-propanol (2.4 g, 8.67 mmol) in THF (25 mL) in an ice bath was added water (7 mL), and then sodium hydroxide (0.416 g, 10.41 mmol) as solid pellets. The mixture was stirred vigorously at the same temperature for 1 h, and diluted with dichloromethane (200 mL). The mixture was washed with 1M sodium hydroxide and brine solution, then dried and concentrated, to provide the title intermediate.

e. Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{(S)-2-hydroxy-3-[4-((R)-tetrahydrofuran-2-carbonyl)piperazin-1-yl]propyl}-8-aza-bicyclo[3.2.1]oct-3-yl)amide

To a solution of endo-N-(8-azabicyclo[3.2.1]oct-3-yl)-3-isopropyl-2-oxo-2,3-dihydrobenzimidazole-1-carboxamide, (prepared as described in Example 1, step (h)) (0.37 g, 1.128 mmol) in ethanol (6 mL) was added 1-[(R)-tetrahydro-2-furoyl]-4-((S)-oxiranyl-methyl)piperazine (0.298 g, 1.241 mmol). The mixture was stirred at 80° C. for 18 h, and concentrated in vacuo, yielding an oily residue. The residue was dissolved in 50% aqueous acetic acid, then purified by preparative HPLC, to provide the title compound (0.15 g) as a TFA salt. (m/z): [M+H]⁺ calcd for C₃₀H₄₄N₆O₅ 569.34; found 569.4. Retention time (anal. HPLC: 10-40% MeCN/H₂O over 6 min)=2.19 min.

Example 11 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{(S)-2-hydroxy-3-[4-((S)-tetrahydrofuran-2-carbonyl)piperazin-1-yl]propyl}-8-aza-bicyclo[3.2.1]oct-3-yl)amide

Using the processes described in Example 10, except in step (a) replacing reagent (R)-tetrahydro-2-furoic acid with (S)-tetrahydro-2-furoic acid, the title compound was prepared (0.133 g) as a TFA salt. The LCMS and analytical HPLC profiles of (S)- and (R)-isomer of Examples 10 and 11 were identical.

Example 12 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(acetylmethylamino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide (by Scheme D)

a. Preparation of N-[(1S,3R,5R)-8-azabicyclo[3.2.1]oct-3-yl]-1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxamide

To a suspension of endo-N-(8-azabicyclo[3.2.1]oct-3-yl)-3-isopropyl-2-oxo-2,3-dihydrobenzimidazole-1-carboxamide TFA salt, (prepared as described in Example 1, step (h)), (15 g, 33.9 mmol) in dichloromethane (500 mL) was added water (500 mL) and enough N,N-diisopropylethylamine (˜20 mL) to bring the aqueous layer to a pH of 8-9. The layers were separated, retaining the organic layer. The aqueous layer was then extracted a second time with dichloromethane (100 mL). The two organic layers were combined and washed with brine. Drying and evaporation of the organic phase provided the free base of the title intermediate (9.7 g, 87% yield) as a yellow powder. (m/z): [M+H]⁺ calcd for C₁₈H₂₄N₄O₂ 329.20; found 329.2. Retention time (anal. HPLC: 2-50% MeCN/H₂O over 6 min)=3.67 min. ¹H NMR (d₆-DMSO): 9.31 (d, 1H), 8.08 (d, 1H), 7.45 (d, 1H), 7.21 (m, 2H), 4.69 (septet, 1H), 4.07 (m, 1H), 3.53 (m, 2H), 1.40-2.00 (m, 8H), 1.48 (d, 6H).

b. Preparation of (S)-1-(benzylmethylamino)-3-chloropropan-2-ol

(S)-Epichlorohydrin (10 mL, 127 mmol) was added to a solution of N-benzyl-methylamine (16.4 mL, 127 mmol) in hexane (60 mL). The mixture was stirred for 16 h at room temperature. Volatiles were removed yielding an oily residue. The product was recovered by column chromatography using SiO₂ methanol/dichloromethane (10:90) with the later eluting peak the product. After drying and filtration, the solvent was removed to provide the title intermediate (19.2 g, 71% yield) as a colorless oil. (m/z): [M+H]⁺ calcd for C₁₁H₁₆ClNO 214.10; found 214.2.

c. Preparation of ((5)-3-chloro-2-hydroxypropyl)methyl-carbamic acid tert-butyl ester

(S)-1-(benzylmethylamino)-3-chloropropan-2-ol (9.1 g, 47.2 mmol) was dissolved in ethyl acetate (75 mL). Boc anhydride (10.1 g, 51.9 mmol) was then added, followed by 10% Pd(OH)₂ on carbon and the mixture placed under 60 psi hydrogen for 16 h. The mixture was filtered through celite, and the volatiles removed yielding an oily residue. The product was recovered by column chromatography. After drying and filtration, the solvent was removed to provide the title intermediate (8.3 g, 87% yield) as a colorless oil.

d. Preparation of methyl-(S)-1-oxiranylmethyl-carbamic acid tert-butyl ester

((S)-3-chloro-2-hydroxypropyl)methyl-carbamic acid tert-butyl ester (3.23 g, 14.4 mmol) was dissolved in 40 mL of a mixture of THF/H₂O (4:1). Sodium hydroxide (0.700 g, 17.3 mmol) was added as a 10 mL solution in water and the reaction was stirred for 16 h at room temperature. The product was taken up in ethyl acetate (200 mL), washed with brine (2×100 mL), dried and filtered to provide the title intermediate (2.5 g, 94% yield) as a colorless oil. (m/z): [M+Na]⁺ calcd for C₉H₁₇NO₃ 188.03; found 210.2. Retention time (anal. HPLC: 2-50% MeCN/H₂O over 6 min)=4.18 min. ¹H NMR (d₆-DMSO): 3.43 (m, 1H), 3.14 (m, 1H), 2.83 (m, 1H), 2.82 (s, 3H), 2.72 (m, 1H), 2.50 (m, 1H), 1.40 (s, 12H).

e. Preparation of ((R)-2-hydroxy-3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydro-benzoimidazole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propyl)methyl-carbamic acid tert-butyl ester

Methyl-(S)-1-oxiranylmethyl-carbamic acid tert-butyl ester (3.1 g, 20.1 mmol) and endo-N-(8-azabicyclo[3.2.1]oct-3-yl)-3-isopropyl-2-oxo-2,3-dihydrobenzimidazole-1-carboxamide, (prepared as described in Example 12, step (a)), (2.2 g, 6.7 mmol) were dissolved in dry ethanol (100 mL) and heated to 80° C. for 16 h. The volatiles were removed yielding an oily residue. The product was recovered by column chromatography to provide the title intermediate (3.0 g, 87% yield) as a light yellow solid. (m/z): [M+H]⁺ calcd for C₂₇H₄₁N₅O₅) 516.32; found 516.5. Retention time (anal. HPLC: 2-50% MeCN/H₂O over 6 min)=4.66 min

f. Preparation of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid [(1S,3R,5R)-8-((S)-2-hydroxy-3-methylamino-propyl)-8-azabicyclo[3.2.1]oct-3-yl]-amide

((R)-2-hydroxy-3-{(1S,3R,5R)-3-[(3-isopropyl-2-oxo-2,3-dihydrobenzoimid-azole-1-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}propyl)methyl-carbamic acid tert-butyl ester (3.0 g, 5.8 mmol) was dissolved in dichloromethane (30 mL) at room temperature and trifluoroacetic acid (25 mL) was added dropwise. After about 20 min the reaction was complete, all volatiles were removed and the oily residue was triturated with ethyl ether. The off-white solids were collected by filtration and dried to provide the title intermediate (3.1 g, 84% yield) as a TFA salt. (m/z): [M+H]⁺ calcd for C₂₂H₃₃N₅O₃ 416.27; found 416.6. Retention time (anal. HPLC: 2-50% MeCN/H₂O over 6 min)=3.17 min.

g. Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(acetylmethylamino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

3-Isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid [(1S,3R,5R)-8-((S)-2-hydroxy-3-methylaminopropyl)-8-azabicyclo[3.2.1]oct-3-yl]amide (3.0 g, 3.9 mmol) was suspended in dichloromethane (10 mL) and cooled to 0° C. N,N-diisopropylethylamine (2.84 mL, 16.3 mmol) was added, followed by acetyl chloride (284 μL, 3.9 mmol). The reaction was allowed to reach room temperature and was complete after about 1 h. The crude reaction mixture was evaporated to give a yellow solid. Further purification via prep HPLC (reverse phase) was accomplished on a gradient of 5-10-60% (5-10% over 10 min; 10-60% over 50 min); flow rate 15 mL/min; detection at 280 nm. The purified fractions were lyophilized to provide the title compound as a TFA salt. A 1:1 mixture of 1N sodium hydroxide and dichloromethane (200 mL) was then added to the lyophilized TFA salt. The organic layer was dried, filtered and evaporated, and lyophilized to provide the title compound (1.05 g, 57% yield) as a free base. (m/z): [M+H]⁺ calcd for C₂₄H₃₅N₅O₄ 458.28; found 458.5. Retention time (anal. HPLC: 2-50% MeCN/H₂O over 6 min)=3.72 min. ¹H NMR (d₆-DMSO): 9.28 (d, 1H), 8.08 (d, 1H), 7.45 (d, 1H), 7.17 (m, 2H), 4.50-4.90 (m, 2H), 4.01 (m, 1H), 3.68 (m, 1H), 3.40-3.60 (m, 1H), 3.33 (s, 1H), 3.23 (m, 2H), 3.02 (s, 2H), 2.83 (s, 2H), 2.24 (m, 2H), 2.11 (m, 2H), 2.03 (s, 3H), 1.80-2.00 (m, 4H), 1.63 (m, 2H), 1.48 (d, 6H).

Example 13 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{(R)-2-hydroxy-3-[methyl-(1-methyl-1H-imidazole-4-sulfonyl)amino]propyl}-8-azabicyclo[3.2.1]oct-3-yl)amide

3-Isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid [(1S,3R,5R)-8-((S)-2-hydroxy-3-methylaminopropyl)-8-azabicyclo[3.2.1]oct-3-yl]amide TFA salt, (prepared as described in Example 12, step (f)), (0.050 g, 0.078 mmol) was suspended in dimethylformamide (5 mL) and cooled to 0° C. N,N-diisopropylethylamine (0.43 mL, 24.6 mmol) was added, followed by 1-methyl-1-H-imidazole sulfonyl chloride (0.017 g, 0.094 mmol). The reaction was allowed to reach room temperature and was judged to be complete after about 1 hr. The reaction was quenched with acetic acid and water (1:1). Volatiles were removed and purification via prep HPLC (reverse phase) was accomplished on a gradient of 15-45% over 50 min; flow rate 20 mL/min, to provide the title compound (0.030 g, 56%) as a TFA salt. (m/z): [M+H]⁺ calcd for C₂₆H₃₇N₇O₅S 560.27; found 560.5. Retention time (anal. HPLC: 2-50% MeCN/H₂O over 4 min)=4.05 min. ¹H NMR (d₆-DMSO): 9.28 (d, 1H), 8.08 (d, 1H), 7.79 (d, 2H), 7.45 (d, 1H), 7.21 (m, 2H), 4.75 (br m, 2H), 4.01 (m, 1H), 3.71 (br s, 4H), 3.23 (m, 3H), 2.88 (m, 1H), 2.77 (s, 2H), 2.29 (m, 2H), 2.08 (m, 2H), 1.93 (m, 4H), 1.59 (m, 2H), 1.48 (d, 6H).

Example 14 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(1-methylureido)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

Using the processes described in Example 13, except replacing 1-methyl-1-H-imidazole sulfonyl chloride with trimethylsilyl isocyanate (0.011 g, 0.094 mmol), the title compound was prepared (0.025 g, 56%) as a TFA salt. (m/z): [M+H]⁺ calcd for C₂₃H₃₄N₆O₄ 459.27; found 459.1. Retention time (anal. HPLC: 10-75% MeCN/H₂O over 4 min)=2.38 min.

Example 15 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{2-hydroxy-3-[methyl-(1-methyl-1H-imidazole-4-sulfonyl)amino]propyl}-8-azabicyclo-[3.2.1]oct-3-yl)amide

Using the processes described in Example 12, steps (a)-(f) and Example 13, except in Example 12, step (b), replacing (S)-epichlorohydrin with racemic epichlorohydrin, the title compound was prepared (0.060 g) as a TFA salt. (m/z): [M+H]⁺ calcd for C₂₆H₃₇N₇O₅S 560.27; found 560.2. Retention time (anal. HPLC: 2-65% MeCN/H₂O over 4 min)=2.76 min.

Example 16 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[2-hydroxy-3-(1-methylureido)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide

Using the processes described in Example 12, steps (a)-(f), Example 13, and Example 14, except in Example 12, step (b), replacing (S)-epichlorohydrin with racemic epichlorohydrin, the title compound was prepared (0.027 g) as a TFA salt. (m/z): [M+H]⁺ calcd for C₂₃H₃₄N₆O₄ 459.27; found 459.2. Retention time (anal. HPLC: 10-75% MeCN/H₂O over 4 min)=2.38 min.

Example 17 Synthesis of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[3-(formylmethylamino)-2-hydroxypropyl]-8-azabicyclo-[3.2.1]oct-3-yl}amide

Using the processes described in Example 12, steps (a)-(f) except replacing (S)-epichlorohydrin with a racemic mixture of epichlorohydrin in Example 12, step (a), 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid [(1S,3R,5R)-8-(-2-hydroxy-3-methylaminopropyl)-8-azabicyclo[3.2.1]oct-3-yl]amide was prepared as a TFA salt.

3-Isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid [(1S,3R,5R)-8-(-2-hydroxy-3-methylaminopropyl)-8-azabicyclo[3.2.1]oct-3-yl]amide (0.050 g, 0.078 mmol) was then suspended in dimethyl-formamide (10 mL) and N,N-diisopropyl-ethylamine (0.43 mL, 24.6 mmol) was added. An excess of ethyl formate (0.59 g, 7.8 mmol) was added and the mixture was heated to 80° C. for 16 h. Volatiles were removed and purification via prep HPLC (reverse phase) was accomplished on a gradient of 15-45% over 50 min; flow rate 20 mL/min to provide the title compound (0.027 g, 61% yield) as a TFA salt. (m/z): [M+H]⁺ calcd for C₂₃H₃₃N₅O₄ 444.26; found 444.2. Retention time (anal. HPLC: 10-75% MeCN/H₂O over 4 min)=2.38 min.

Example 18 Synthesis of Acid Salts of 3-isopropyl-2-oxo-2,3-dihydrobenzo-imidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(acetylmethylamino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide Example 18-1 Synthesis of a Hydrochloride Salt

To a mixture of lyophilized 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(acetylmethyl-amino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide, the product of Example 12, (20 mg) dispersed in 1 mL of deionized water was added 4 μL of concentrated hydrochloric acid. The mixture was heated to 50° C. and 180 μL of methanol was added (until complete dissolution). The solution was filtered with a 0.2 micron syringe filter, capped in a clean vial and cooled to 4° C. The vial was allowed to equilibrate to room temperature and uncapped to slowly evaporate the excess solvent overnight. The following morning large needle-like crystals and star-burst shaped crystal clusters were recovered.

The powder x-ray diffraction pattern of the product, shown in FIG. 1 was obtained with a Rigaku diffractometer using Cu Kα (30.0 kV, 15.0 mA) radiation. The analysis was performed with the goniometer running in continuous-scan mode of 2° per min with a step size of 0.03° over a range of 2 to 40°. Samples were prepared on quartz specimen holders as a thin layer of powdered material. The instrument was calibrated with a silicon metal standard.

As shown in FIG. 1, the spectrum comprises prominent peaks at 2θ values of 6.17, 15.68, 17.1, 18.68, 21.86, 25.01, 28.16, 31.48, and 37.97 degrees.

Example 18-2 Alternate Synthesis of a Hydrochloride Salt

Amorphous powder of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(acetylmethyl-amino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide, the product of Example 12, (20 mg) dispersed in methanol (1 mL) was compounded as a stock free base solution (Solution A, 0.044M), and filtered through a 0.2 micron syringe filter.

A stock solution (Solution B, concentration 0.046M) was prepared from concentrated hydrochloric acid (40 μL) and methanol (10 mL).

Crystallization screening was performed by mixing a 20 μL drop of Solution A with varying volumes of Solution B (0, 10, 20, and 40 μL; corresponding to 0, 0.5, 1.0, and 2.0 mole equivalents of acid respectively) and allowing each mixture to evaporate slowly in an open vial overnight. The following day, the solids remaining on the wall or base of each vial were observed using polarized light microscopy (an Olympus SZX stereozoom microscope with cross-polarizing filters).

Birefringent particles, mostly needle-shaped crystals or polycrystalline clusters, were observed in the solids which resided on the walls or base of the glass vials of the 0.5 and 2.0 mole hydrochloric acid equivalent vials.

The test vial with 0 μL of solution B was a control experiment to determine crystallinity of the free base. No crystalline or birefringent free base solids were observed in the solids which resided on the walls or base of the free base glass vial even after a period of several weeks.

Example 18-3 Through 18-6 Synthesis of Other Acid Salts

Following the procedure of Example 18-2, crystalline forms of acid salts of 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(acetylmethylamino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide were prepared using the indicated equivalents of acid:

Example 18-3 hydrobromic acid (0.5, 1.0, and 1.5 M);

Example 18-4 phosphoric acid (0.25, 0.75, and 1.5 M);

Example 18-5 sulfuric acid (0.25, 0.75, and 1.5 M); and

Example 18-6 nitric acid (0.5, 1.0, and 2.0 M).

Examples 19-37

Using processes similar to those described above, the compounds of Examples 19-37 were prepared.

Calc. Obs. # —NR³R⁴ * Forumla [M + H]⁺ [M + H]⁺ 19

— C₂₉H₄₄N₆O₄ 541.34 541.4 20

— C₂₇H₄₁N₅O₅S 548.28 548.2 21

— C₂₇H₄₂N₆O₅S 563.29 563.2 22

— C₂₈H₄₂N₆O₄ 527.33 527.2 23

— C₂₈H₃₆N₆O₄ 521.28 521.2 24

— C₂₄H₃₅N₅O₄ 458.27 458.2 25

— C₂₃H₃₅N₅O₅S 494.24 494.5 26

— C₂₅H₃₇N₅O₅ 488.28 488.2 27

R C₂₅H₃₇N₅O₅ 488.28 488.2 28

R C₂₃H₃₃N₅O₄ 444.25 444.2 29

R C₂₃H₃₅N₅O₅S 494.25 494.2 30

R C₂₈H₃₆N₆O₄ 521.28 521.2 31

— C₂₈H₄₂N₆O₅S 575.29 575.3 32

S C₂₈H₃₆N₆O₄ 521.28 521.2 33

S C₂₅H₃₇N₅O₅ 488.28 488.2 34

S C₂₃H₃₃N₅O₄ 444.25 444.2 35

S C₂₃H₃₅N₅O₅S 494.24 494.2 36

S C₂₆H₃₇N₇O₅S 560.26 560.2 37

S C₂₄H₃₅N₅O₄ 458.27 458.2

Assay 1: Radioligand Binding Assay on 5-HT_(4(c)) Human Receptors

a. Membrane Preparation 5-HT_(4(c))

HEK-293 (human embryonic kidney) cells stably-transfected with human 5-HT_(4(c)) receptor cDNA (Bmax=˜6.0 pmol/mg protein, as determined using [³H]-GR113808 membrane radioligand binding assay) were grown in T-225 flasks in Dulbecco's Modified Eagles Medium (DMEM) containing 4,500 mg/L D-glucose and pyridoxine hydrochloride (GIBCO-Invitrogen Corp., Carlsbad Calif.: Cat #11965) supplemented with 10% fetal bovine serum (FBS) (GIBCO-Invitrogen Corp.: Cat #10437), 2 mM L-glutamine and (100 units) penicillin-(100 μg) streptomycin/ml (GIBCO-Invitrogen Corp.: Cat #15140) in a 5% CO₂, humidified incubator at 37° C. Cells were grown under continuous selection pressure by the addition of 800 μg/mL geneticin (GIBCO-Invitrogen Corp.: Cat #10131) to the medium.

Cells were grown to roughly 60-80% confluency (<35 subculture passages). At 20-22 hours prior to harvesting, cells were washed twice and fed with serum-free DMEM. All steps of the membrane preparation were performed on ice. The cell monolayer was lifted by gentle mechanical agitation and trituration with a 25 mL pipette. Cells were collected by centrifugation at 1000 rpm (5 min).

For the membrane preparation, cell pellets were resuspended in ice-cold 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), pH 7.4 (membrane preparation buffer) (40 mL/total cell yield from 30-40 T225 flasks) and homogenized using a polytron disrupter (setting 19, 2×10 s) on ice. The resultant homogenates were centrifuged at 1200 g for 5 min at 4° C. The pellet was discarded and the supernatant centrifuged at 40,000 g (20 min). The pellet was washed once by resuspension with membrane preparation buffer and centrifugation at 40,000 g (20 min). The final pellet was resuspended in 50 mM HEPES, pH 7.4 (assay buffer) (equivalent 1 T225 flask/1 mL). Protein concentration of the membrane suspension was determined by the method of Bradford (Bradford, 1976). Membranes were stored frozen in aliquots at −80° C.

b. Radioligand Binding Assays

Radioligand binding assays were performed in 1.1 mL 96-deep well polypropylene assay plates (Axygen) in a total assay volume of 400 μL containing 2 μg membrane protein in 50 mM HEPES pH 7.4, containing 0.025% bovine serum albumin (BSA). Saturation binding studies for determination of K_(d) values of the radioligand were performed using [³H]-GR113808 (Amersham Inc., Bucks, UK: Cat #TRK944; specific activity ˜82 Ci/mmol) at 8-12 different concentrations ranging from 0.001 nM -5.0 nM. Displacement assays for determination of pK_(i) values of compounds were performed with [³H]-GR113808 at 0.15 nM and eleven different concentrations of compound ranging from 10 pM -100 μM.

Test compounds were received as 10 mM stock solutions in DMSO and diluted to 400 μM into 50 mM HEPES pH 7.4 at 25° C., containing 0.1% BSA, and serial dilutions (1:5) then made in the same buffer. Non-specific binding was determined in the presence of 1 μM unlabeled GR113808. Assays were incubated for 60 min at room temperature, and then the binding reactions were terminated by rapid filtration over 96-well GF/B glass fiber filter plates (Packard BioScience Co., Meriden, Conn.) presoaked in 0.3% polyethyleneimine. Filter plates were washed three times with filtration buffer (ice-cold 50 mM HEPES, pH7.4) to remove unbound radioactivity. Plates were dried, 35 μL Microscint-20 liquid scintillation fluid (Packard BioScience Co., Meriden, Conn.) was added to each well and plates were counted in a Packard Topcount liquid scintillation counter (Packard BioScience Co., Meriden, Conn.).

Binding data were analyzed by nonlinear regression analysis with the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, Calif.) using the 3-parameter model for one-site competition. The BOTTOM (curve minimum) was fixed to the value for nonspecific binding, as determined in the presence of 1 μM GR113808. K_(i) values for test compounds were calculated, in Prism, from the best-fit IC₅₀ values, and the K_(d) value of the radioligand, using the Cheng-Prusoff equation (Cheng and Prusoff, Biochemical Pharmacology, 1973, 22, 3099-108): K_(i)=IC₅₀/(1+[L]/K_(d)) where [L]=concentration [³H]-GR113808. Results are expressed as the negative decadic logarithm of the K_(i) values, pK_(i).

Test compounds having a higher pK_(i) value in this assay have a higher binding affinity for the 5-HT₄ receptor. The compounds of the invention which were tested in this assay had a pK_(i) value ranging from about 6.7 to about 8.3, typically ranging from about 7.0 to about 8.0.

Assay 2: Radioligand Binding Assay on 5-HT_(3A) Human Receptors: Determination of Receptor Subtype Selectivity

a. Membrane Preparation 5-HT_(3A)

HEK-293 (human embryonic kidney) cells stably-transfected with human 5-HT_(3A) receptor cDNA were obtained from Dr. Michael Bruess (University of Bonn, GDR) (Bmax=˜9.0 pmol/mg protein, as determined using [³H]-GR65630 membrane radioligand binding assay). Cells were grown in T-225 flasks or cell factories in 50% Dulbecco's Modified Eagles Medium (DMEM) (GIBCO-Invitrogen Corp., Carlsbad, Calif.: Cat #11965) and 50% Ham's F12 (GIBCO-Invitrogen Corp.: Cat #11765) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT: Cat #SH30070.03) and (50 units) penicillin-(50 μg) streptomycin/ml (GIBCO-Invitrogen Corp.: Cat #15140) in a 5% CO₂, humidified incubator at 37° C.

Cells were grown to roughly 70-80% confluency (<35 subculture passages). All steps of the membrane preparation were performed on ice. To harvest the cells, the media was aspirated and cells were rinsed with Ca²⁺, Mg²⁺-free Dulbecco's phosphate buffered saline (dPBS). The cell monolayer was lifted by gentle mechanical agitation. Cells were collected by centrifugation at 1000 rpm (5 min). Subsequent steps of the membrane preparation followed the protocol described above for the membranes expressing 5-HT_(4(c)) receptors.

b. Radioligand Binding Assays

Radioligand binding assays were performed in 96-well polypropylene assay plates in a total assay volume of 200 μL containing 1.5-2 μg membrane protein in 50 mM HEPES pH 7.4, containing 0.025% BSA assay buffer. Saturation binding studies for determination of K_(d) values of the radioligand were performed using [³H]-GR65630 (PerkinElmer Life Sciences Inc., Boston, Mass.: Cat #NET1011, specific activity ˜85 Ci/mmol) at twelve different concentrations ranging from 0.005 nM to 20 nM. Displacement assays for determination of pK_(i) values of compounds were performed with [³H]-GR65630 at 0.50 nM and eleven different concentrations of compound ranging from 10 pM to 100 μM. Compounds were received as 10 mM stock solutions in DMSO (see section 3.1), diluted to 400 μM into 50 mM HEPES pH 7.4 at 25° C., containing 0.1% BSA, and serial (1:5) dilutions then made in the same buffer. Non-specific binding was determined in the presence of 10 μM unlabeled MDL72222. Assays were incubated for 60 min at room temperature, then the binding reactions were terminated by rapid filtration over 96-well GF/B glass fiber filter plates (Packard BioScience Co., Meriden, Conn.) presoaked in 0.3% polyethyleneimine. Filter plates were washed three times with filtration buffer (ice-cold 50 mM HEPES, pH7.4) to remove unbound radioactivity. Plates were dried, 35 μL Microscint-20 liquid scintillation fluid (Packard BioScience Co., Meriden, Conn.) was added to each well and plates were counted in a Packard Topcount liquid scintillation counter (Packard BioScience Co., Meriden, Conn.).

Binding data were analyzed using the non-linear regression procedure described above to determine K_(i) values. The BOTTOM (curve minimum) was fixed to the value for nonspecific binding, as determined in the presence of 10 μM MDL72222. The quantity [L] in the Cheng-Prusoff equation was defined as the concentration [³H]-GR65630.

Selectivity for the 5-HT₄ receptor subtype with respect to the 5-HT₃ receptor subtype was calculated as the ratio K_(i)(5-HT_(3A))/K_(i)(5-HT_(4(c))). The compounds of the invention which were tested in this assay had a 5-HT₄/5-HT₃ receptor subtype selectivity ranging from about 20 to about 5600, typically ranging from about 100 to about 1700.

Assay 3: Whole-cell cAMP Accumulation Flashplate Assay with HEK-293 Cells Expressing Human 5-HT_(4(c)) Receptors

In this assay, the functional potency of a test compound was determined by measuring the amount of cyclic AMP produced when HEK-293 cells expressing 5-HT₄ receptors were contacted with different concentrations of test compound.

a. Cell Culture

HEK-293 (human embryonic kidney) cells stably-transfected with cloned human 5-HT_(4(c)) receptor cDNA were prepared expressing the receptor at two different densities: (1) at a density of about 0.5-0.6 pmol/mg protein, as determined using a [³H]-GR113808 membrane radioligand binding assay, and (2) at a density of about 6.0 pmol/mg protein. The cells were grown in T-225 flasks in Dulbecco's Modified Eagles Medium (DMEM) containing 4,500 mg/L D-glucose (GIBCO-Invitrogen Corp.: Cat #11965) supplemented with 10% fetal bovine serum (FBS) (GIBCO-Invitrogen Corp.: Cat #10437) and (100 units) penicillin-(100 μg) streptomycin/ml (GIBCO-Invitrogen Corp.: Cat #15140) in a 5% CO₂, humidified incubator at 37° C. Cells were grown under continuous selection pressure by the addition of geneticin (800 μg/mL: GIBCO-Invitrogen Corp.: Cat #10131) to the medium.

b. Cell Preparation

Cells were grown to roughly 60-80% confluency. Twenty to twenty-two hours prior to assay, cells were washed twice, and fed, with serum-free DMEM containing 4,500 mg/L D-glucose (GIBCO-Invitrogen Corp.: Cat #11965). To harvest the cells, the media was aspirated and 10 mL Versene (GIBCO-Invitrogen Corp.: Cat #15040) was added to each T-225 flask. Cells were incubated for 5 min at RT and then dislodged from the flask by mechanical agitation. The cell suspension was transferred to a centrifuge tube containing an equal volume of pre-warmed (37° C.) dPBS and centrifuged for 5 min at 1000 rpm. The supernatant was discarded and the pellet was re-suspended in pre-warmed (37° C.) stimulation buffer (10 mL equivalent per 2-3 T-225 flasks). This time was noted and marked as time zero. The cells were counted with a Coulter counter (count above 8 μm, flask yield was 1-2×10⁷ cells/flask). Cells were resuspended at a concentration of 5×10⁵ cells/ml in pre-warmed (37° C.) stimulation buffer (as provided in the flashplate kit) and preincubated at 37° C. for 10 min.

cAMP assays were performed in a radioimmunoassay format using the Flashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (SMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), according to the manufacturer's instructions.

Cells were grown and prepared as described above. Final cell concentrations in the assay were 25×10³ cells/well and the final assay volume was 100 μL. Test compounds were received as 10 mM stock solutions in DMSO, diluted to 400 μM into 50 mM HEPES pH 7.4 at 25° C., containing 0.1% BSA, and serial (1:5) dilutions then made in the same buffer. Cyclic AMP accumulation assays were performed with 11 different concentrations of compound ranging from 10 pM to 100 μM (final assay concentrations). A 5-HT concentration-response curve (10 pM to 100 μM) was included on every plate. The cells were incubated, with shaking, at 37° C. for 15 min and the reaction terminated by addition of 100 μl of ice-cold detection buffer (as provided in the flashplate kit) to each well. The plates were sealed and incubated at 4° C. overnight. Bound radioactivity was quantified by scintillation proximity spectroscopy using the Topcount (Packard BioScience Co., Meriden, Conn.).

The amount of cAMP produced per mL of reaction was extrapolated from the cAMP standard curve, according to the instructions provided in the manufacturer's user manual. Data were analyzed by nonlinear regression analysis with the GraphPad Prism Software package using the 3-parameter sigmoidal dose-response model (slope constrained to unity). Potency data are reported as pEC₅₀ values, the negative decadic logarithm of the EC₅₀ value, where EC₅₀ is the effective concentration for a 50% maximal response.

Test compounds exhibiting a higher pEC₅₀ value in this assay have a higher potency for agonizing the 5-HT₄ receptor. The compounds of the invention which were tested in this assay, for example, in the cell line (1) having a density of about 0.5-0.6 pmol/mg protein, had a pEC₅₀ value ranging from about 7.5 to about 9.0, typically ranging from about 7.7 to about 8.5.

Assay 4: In vitro Voltage Clamp Assay of Inhibition of Potassium Ion Current in Whole Cells Expressing the hERG Cardiac Potassium Channel

CHO-K1 cells stably transfected with hERG cDNA were obtained from Gail Robertson at the University of Wisconsin. Cells were held in cryogenic storage until needed. Cells were expanded and passaged in Dulbecco's Modified Eagles Medium/F12 supplemented with 10% fetal bovine serum and 200 μg/mL geneticin. Cells were seeded onto poly-D-lysine (100 μg/mL) coated glass coverslips, in 35 mm² dishes (containing 2 mL medium) at a density that enabled isolated cells to be selected for whole cell voltage-clamp studies. The dishes were maintained in a humidified, 5% CO₂ environment at 37° C.

Extracellular solution was prepared at least every 7 days and stored at 4° C. when not in use. The extracellular solution contained (mM): NaCl (137), KCl (4), CaCl₂ (1.8), MgCl₂ (1), Glucose (10), 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) (10), pH 7.4 with NaOH. The extracellular solution, in the absence or presence of test compound, was contained in reservoirs, from which it flowed into the recording chamber at approximately 0.5 mL/min. The intracellular solution was prepared, aliquoted and stored at −20° C. until the day of use. The intracellular solution contained (mM): KCl (130), MgCl₂ (1), ethylene glycol-bis(beta-aminoethyl ether) N,N,N′,N′-tetra acetic acid salt (EGTA) (5), MgATP (5), 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) (10), pH 7.2 with KOH. All experiments were performed at room temperature (20-22° C.).

The coverslips on which the cells were seeded were transferred to a recording chamber and perfused continuously. Gigaohm seals were formed between the cell and the patch electrode. Once a stable patch was achieved, recording commenced in the voltage clamp mode, with the initial holding potential at −80 mV. After a stable whole-cell current was achieved, the cells were exposed to test compound. The standard voltage protocol was: step from the holding potential of −80 mV to +20 mV for 4.8 sec, repolarize to −50 mV for 5 sec and then return to the original holding potential (−80 mV). This voltage protocol was run once every 15 sec (0.067 Hz). Peak current amplitudes during the repolarization phase were determined using pClamp software. Test compounds at a concentration of 3 μM were perfused over the cells for 5 minutes, followed by a 5-minute washout period in the absence of compound. Finally a positive control (cisapride, 20 nM) was added to the perfusate to test the function of the cell. The step from −80 mV to +20 mV activates the hERG channel, resulting in an outward current. The step back to −50 mV results in an outward tail current, as the channel recovers from inactivation and deactivates.

Peak current amplitudes during the repolarization phase were determined using pCLAMP software. The control and test article data were exported to Origin® (OriginLab Corp., Northampton Mass.) where the individual current amplitudes were normalized to the initial current amplitude in the absence of compound. The normalized current means and standard errors for each condition were calculated and plotted versus the time course of the experiment.

Comparisons were made between the observed K⁺ current inhibitions after the five-minute exposure to either the test article or vehicle control (usually 0.3% DMSO). Statistical comparisons between experimental groups were performed using a two-population, independent t-test (Microcal Origin v. 6.0). Differences were considered significant at p<0.05.

The smaller the percentage inhibition of the potassium ion current in this assay, the smaller the potential for test compounds to change the pattern of cardiac repolarization when used as therapeutic agents. The compounds of the invention which were tested in this assay at a concentration of 3 μM typically exhibited an inhibition of the potassium ion current of less than about 20%, more typically, less than about 15%.

Assay 5: In vitro Model of Oral Bioavailability: Caco-2 Permeation Assay

The Caco-2 permeation assay was performed to model the ability of test compounds to pass through the intestine and get into the blood stream after oral administration. The rate at which test compounds in solution permeate a cell monolayer designed to mimic the tight junction of human small intestinal monolayers was determined.

Caco-2 (colon, adenocarcinoma; human) cells were obtained from ATCC (American Type Culture Collection; Rockville, Md.). For the permeation study, cells were seeded at a density of 63,000 cells/cm² on pre-wetted transwells polycarbonate filters (Costar; Cambridge, Mass.). A cell monolayer was formed after 21 days in culture. Following cell culture in the transwell plate, the membrane containing the cell monolayer was detached from the transwell plate and inserted into the diffusion chamber (Costar; Cambridge, Mass.). The diffusion chamber was inserted into the heating block which was equipped with circulating external, thermostatically regulated 37° C. water for temperature control. The air manifold delivered 95% O₂/5% CO₂ to each half of a diffusion chamber and created a laminar flow pattern across the cell monolayer, which was effective in reducing the unstirred boundary layer.

The permeation study was performed with test compound concentrations at 100 μM and with ¹⁴C-mannitol to monitor the integrity of the monolayer. All experiments were conducted at 37° C. for 60 min. Samples were taken at 0, 30 and 60 min from both the donor and receiver sides of the chamber. Samples were analyzed by HPLC or liquid scintillation counting for test compound and mannitol concentrations. The permeation coefficient (K_(p)) in cm/sec was calculated.

In this assay, a K_(p) value greater than about 10×10⁻⁶ cm/sec is considered indicative of favorable bioavailability. The compounds of the invention that were tested in this assay typically exhibited K_(p) values of between about 5×10⁻⁶ cm/sec and about 55×10⁻⁶ cm/sec, more typically between about 20×10⁻⁶ cm/sec and about 40×10⁻⁶ cm/sec.

Assay 6: Pharmacokinetic Study in the Rat

Aqueous solution formulations of test compounds were prepared in 0.1% lactic acid at a pH of between about 5 and about 6. Male Sprague-Dawley rats (CD strain, Charles River Laboratories, Wilmington, Mass.) were dosed with test compounds via intravenous administration (IV) at a dose of 2.5 mg/kg or by oral gavage (PO) at a dose of 5 mg/kg. The dosing volume was 1 mL/kg for IV and 2 mL/kg for PO administration. Serial blood samples were collected from animals pre-dose, and at 2 (IV only), 5, 15, and 30 min, and at 1, 2, 4, 8, and 24 hours post-dose. Concentrations of test compounds in blood plasma were determined by liquid chromatography-mass spectrometry analysis (LC-MS/MS) (MDS SCIEX, API 4000, Applied Biosystems, Foster City, Calif.) with a lower limit of quantitation of 1 ng/mL.

Standard pharmacokinetic parameters were assessed by non-compartmental analysis (Model 201 for IV and Model 200 for PO) using WinNonlin (Version 4.0.1, Pharsight, Mountain View, Calif.). The maximum in the curve of test compound concentration in blood plasma vs. time is denoted C_(max). The area under the concentration vs. time curve from the time of dosing to the last measurable concentration (AUC(0-t)) was calculated by the linear trapezoidal rule. Oral bioavailability (F(%)), i.e. the dose-normalized ratio of AUC(0-t) for PO administration to AUC(0-t) for IV administration, can be calculated as: F(%)=AUC _(PO) /AUC _(IV)×Dose_(IV)/Dose_(PO)×100%

Test compounds which exhibit larger values of the parameters C_(max), AUC(0-t), and F(%) in this assay are expected to have greater bioavailability when administered orally. The compounds of the invention that were tested in this assay had C_(max) values typically ranging from about 0.05 to about 0.47 μg/mL and AUC(0-t) values typically ranging from about 0.1 to about 1.2 μg·hr/mL. By way of example, the compound of Example 2 had a C_(max) value of 0.11 μg/mL, and an AUC(0-t) value of 0.54 μg·hr/mL.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Additionally, all publications, patents, and patent documents cited hereinabove are incorporated by reference herein in full, as though individually incorporated by reference. 

1. A compound of formula (I):

wherein: R¹ is hydrogen or C₁₋₃alkyl; R² is hydrogen or C₁₋₃alkyl; R³ is C₁₋₃alkyl; R⁴ is —C(O)R⁵, —S(O)₂R⁶,

or R³ and R⁴ together with the nitrogen atom to which they are attached form

R⁵ is hydrogen, C₁₋₃alkyl, —NH₂, or pyridinyl, wherein C₁₋₃alkyl is optionally substituted with hydroxy; R⁶ is C₁₋₃alkyl, —NH₂, or imidazolyl, wherein imidazolyl is optionally substituted with C₁₋₃alkyl; R⁷ is —NR⁹S(O)₂C₁₋₃alkyl, —NR¹⁰C(O)R¹¹, or

R⁸ is —S(O)₂C₁₋₃alkyl or —C(O)R¹²; R⁹, R¹⁰, and R¹¹ are each independently C₁₋₃alkyl; and R¹² is hydrogen, C₁₋₃alkyl, or tetrahydrofuranyl; or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof.
 2. The compound of claim 1, wherein R¹ is ethyl or isopropyl; and R² is hydrogen.
 3. The compound of claim 2, wherein R³ is methyl.
 4. The compound of claim 1, wherein R⁴ is —C(O)R⁵.
 5. The compound of claim 4, wherein R⁵ is hydrogen or C₁₋₃alkyl.
 6. The compound of claim 1, wherein R⁴ is —S(O)₂R⁶.
 7. The compound of claim 6, wherein R⁶ is methyl or 1-methylimidazol-4-yl.
 8. The compound of claim 1, wherein R⁴ is


9. The compound of claim 1, wherein R³ and R⁴ together with the nitrogen atom to which they are attached form


10. The compound of claim 9, wherein R⁸ is —S(O)₂CH₃, —C(O)CH₃, or —C(O)-tetrahydrofuran-2-yl.
 11. The compound of claim 1, wherein the compound is selected from: 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo-[3.2.1]oct-3-yl}amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{2-hydroxy-3-[4-(tetrahydrofuran-2-carbonyl)piperazin-1-yl]propyl}-8-azabicyclo-[3.2.1]oct-3-yl)amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[3-(4-acetylpiperazin-1-yl)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide; 3 -isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{3-[(1,1-dioxotetrahydro-1λ⁶-thiophen-3-yl)methylamino]-2-hydroxypropyl}-8-aza-bicyclo[3.2.1]oct-3-yl)amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(S)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(S)-3-(4-acetylpiperazin-1-yl)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(4-acetylpiperazin-1-yl)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{(S)-2-hydroxy-3-[4-((R)-tetrahydrofuran-2-carbonyl)piperazin-1-yl]propyl}-8-aza-bicyclo[3.2.1]oct-3-yl)amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{(S)-2-hydroxy-3-[4-((S)-tetrahydrofuran-2-carbonyl)piperazin-1-yl]propyl}-8-aza-bicyclo[3.2.1]oct-3-yl)amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(acetylmethylamino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{(R)-2-hydroxy-3-[methyl-(1-methyl-1H-imidazole-4-sulfonyl)amino]propyl}-8-azabicyclo[3.2.1]oct-3-yl)amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(1-methylureido)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid ((1S,3R,5R)-8-{2-hydroxy-3-[methyl-(1-methyl-1H-imidazole-4-sulfonyl)amino]propyl}-8-azabicyclo-[3.2.1]oct-3-yl)amide; 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[2-hydroxy-3-(1-methylureido)propyl]-8-aza-bicyclo[3.2.1]oct-3-yl}amide; and 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[3-(formylmethylamino)-2-hydroxypropyl]-8-azabicyclo-[3.2.1]oct-3-yl}amide; or a pharmaceutically-acceptable salt thereof.
 12. The compound of claim 1, wherein the compound is 3-isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-carboxylic acid {(1S,3R,5R)-8-[(R)-3-(acetylmethylamino)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide; or a pharmaceutically-acceptable salt thereof.
 13. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.
 14. A process for preparing a compound of formula (I):

wherein R¹, R², R³, and R⁴ are as defined in claim 1, or a salt or stereoisomer thereof, the process comprising: (a) reacting a compound of formula (III):

with a compound of the formula (IV):

or (b) reacting a compound of formula (V):

with a compound of formula (VI):

or (c) reacting a compound of formula (VIII):

with a compound of formula (IX): L¹-R⁴  (IX) wherein L¹ is a leaving group and R⁴ is as defined herein; or (d) reacting a compound of formula (VIII) with O═C═NP¹, wherein P¹ is an amino-protecting group, and then removing the amino-protecting group P¹; or (e) when R² is hydrogen, reacting a compound of formula (VI) with a compound of formula (VII):

(f) when R² is hydrogen, reacting a compound of formula (VI) with a compound of formula (VII′):

wherein L² is a leaving group; to provide a compound of formula (I), or a salt or stereoisomer thereof.
 15. A compound of formula (VIII):

wherein R¹ is hydrogen or C₁₋₃alkyl; R² is hydrogen or C₁₋₃alkyl; and R³ is C₁₋₃alkyl; or a salt or stereoisomer or protected derivative thereof. 